Transposon end compositions and methods for modifying nucleic acids

ABSTRACT

The present invention provides methods, compositions and kits for using a transposase and a transposon end for generating extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro, then using a DNA polymerase for generating 5′- and 3′-tagged single-stranded DNA fragments without performing a PCR amplification reaction, wherein the first tag on the 5′-ends exhibits the sequence of the transferred transposon end and optionally, an additional arbitrary sequence, and the second tag on the 3′-ends exhibits a different sequence from the sequence exhibited by the first tag. The method is useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including processes for metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and comparative genomic sequencing (CGS), including massively parallel DNA sequencing (so-called “next-generation sequencing.).

This application claims priority to U.S. Provisional Application Ser.No. 61/108,321, filed Oct. 24, 2008; 61/108,326, filed Oct. 24, 2008;61/108,329, filed Oct. 24, 2008; 61/155,431, filed Feb. 25, 2009; and61/184,530, filed Jun. 5, 2009, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods, compositions and kits forusing transposase and a transposon end compositions for generating alibrary of tagged DNA fragments from target DNA. The ssDNA fragmentsgenerated are useful as templates, e.g., for a variety of applications,including, e.g., high throughput, massively parallel and/or multiplexDNA sequencing.

BACKGROUND OF THE INVENTION

There are a variety of methods and applications for which it isdesirable to generate a library of fragmented and tagged DNA moleculesfrom double-stranded DNA (dsDNA) target molecules. Often, the purpose isto generate smaller, single-stranded DNA (ssDNA) molecules (e.g., DNAfragments) from larger dsDNA molecules for use as templates in DNA orRNA polymerase reactions (e.g., for use as templates in DNA sequencingreactions or in DNA or RNA amplification reactions in which a primeranneals to the tag and is extended by a polymerase).

Until recently, most DNA sequencing was performed using the Sangerdideoxy chain termination sequencing method, in which a primer isextended by a polymerase using the DNA to be sequenced as a template.Four reactions are conducted, each with a mixture of all canonicalnucleotides (dATP, dCTP, dGTP, and dTTP) and one of the fourchain-terminating dideoxynucleotide (ddATP, ddCTP, ddGTP, or ddTTP) andeach reaction produces a nested set of chain terminated fragments thatbegin with the primer and terminate with the dideoxynucleotide. Whenthese chain-terminated DNA molecules are separated by size followingelectrophoresis, the order in which the ddNTPs were incorporatedreflects the sequence of the template DNA. Using these methods, thesequence could be determined for a few hundred or a thousand bases fromthe primer site. Determination of larger sequences required piecing thelarger sequence together from overlapping information from numerousclones.

Because these traditional methods require large amounts of DNA template,and because these methods produced poor results if large amounts ofnon-template DNA is present, Sanger dideoxy sequencing is oftenperformed using a cloned or amplified DNA. For example, most of thesequencing carried out during the Human Genome Project, which formallybegan in 1990 and culminated with the announcement of the completion ofa ‘rough draft’ of the human genome sequence in 2000 and publication ofthe sequence of the last human chromosome in 2006, was based on usinggenomic libraries consisting of a population of host bacteria, each ofwhich carried a DNA molecule that was cloned into a DNA vector, suchthat the collection of all DNA clones, each carrying a piece of thegenomic DNA, represented the entire genome. This was a tedious andhighly iterative process, involving construction and banking of largenumbers of DNA clones (e.g., BAC clones), which, in turn, were oftensubcloned to generate libraries of smaller DNA clones, which were usedas sequencing templates. Often, the primers used in these methods weredesigned to anneal to the vector such that they would be extended intothe unknown cloned DNA during the sequencing reactions. This approachallowed the same set of primers to be used for analyzing many differentclones.

In order to decrease the amount of subcloning required for the humangenome sequencing project, one method that was sometimes used was “invitro transposition.” The in vitro transposition method comprises usingmobile genetic elements called transposons to insert a small piece ofDNA of known sequence into the middle of the unknown DNA. The methodcomprises incubating a DNA clone from a genomic library with atransposon under conditions wherein a single insertion of the transposoninto the DNA clone occurs, then transforming E. coli cells with analiquot of the in vitro transposition reaction, and selecting cells thatcontained a marker, such as an antibiotic resistance marker, encoded bythe transposon. Thus, the in vitro transposition reaction generates alibrary of “transposon insertion clones” from the parent DNA clone, eachof which contains the transposon inserted at a different location in theDNA clone. Each insertion clone is then sequenced outward from each endof the transposon using a different primer for each DNA strand. Asdescribed above, the complete sequence of the parent DNA clone isconstructed by overlapping the sequences obtained from differentinsertion clones. Examples of the use of this transposon insertionmethod for the Human Genome Project were described by Butterfield, Y S Net al., Nucleic Acids Res 30: 2460-2468, 2002; Shevchenko, Y et al.,Nucleic Acids Res 30: 2469-2477, 2002; and Haapa, S et al., Genome Res9: 308-315, 1999. Use of the in vitro transposition process for theHuman Genome Project facilitated the complete sequencing of both genomicDNA clones and clones of cDNA generated from mRNA encoded by the genomicDNA. However, one disadvantage of this in vitro transposition method wasthat it was not totally in vitro, since it required the steps oftransforming E. coli cells, selecting E. coli colonies that containedtransposon insertions, and then isolating the DNA from the transposoninsertion clones for sequencing.

In order to eliminate the requirement to transform E. coli cells with analiquot of the in vitro transposition reaction and culture the E. colicells on selective medium to obtain transposon insertion clones,Teknanen et al. (U.S. Pat. No. 6,593,113) developed totally in vitrotransposon-based methods comprising an in vitro transposition reactionand a PCR amplification reaction to select sequencing templates.According to Teknanen et al., the examined DNA or target DNA used intheir methods can range from a few base pairs to up to 40 kilobasepairs, with the only limiting factor for not using even longer DNAsegments as target DNA being the inability of amplification reactions,such as PCR, to amplify longer segments. Thus, in some embodiments forgenerating sequencing templates using this method, the examined DNA ortarget DNA of up to about 40 Kb is first subjected to an in vitrotransposition reaction, and then is PCR amplified using, as a first PCRprimer, a fixed primer that is complementary to a known sequence in thetarget DNA or, if the target DNA is cloned in a vector, a fixed primerthat is complementary to a sequence in the vector, and as a second PCRprimer, a selective primer that is complementary to a sequence of thetransposon end to which the target DNA is joined, plus, optionally, oneto ten additional nucleotides of known identity at its 3′ end. Inanother embodiment, two selective primers are used for the PCRamplification step, at least one of which has one to ten additionalnucleotides of known identity at its 3′ end. The methods of Teknanen etal. provide certain benefits for Sanger sequencing because theyeliminate the need to use E. coli cells to select DNA molecules thathave transposon insertions. However, these methods are limited to targetDNA of a size up to about 40 Kb and, due to the use of fixed orselective primers, the methods select for DNA molecules that exhibitonly a portion of the sequences exhibited by the target DNA. Therefore,although these methods were useful for Sanger sequencing, they are notsuitable for generating sequencing templates for the newer“next-generation” DNA sequencing methods, which are capable ofgenerating sequence data from up to millions of sequencing templates ina single sequencing run using a massively parallel or multiplex format.

Next-generation sequencing platforms include the 454 FLX™ or 454TITANIUM™ (Roche), the SOLEXA™ Genome Analyzer (Illumina), theHELISCOPE™ Single Molecule Sequencer (Helicos Biosciences), and theSOLID™ DNA Sequencer (Life Technologies/Applied Biosystems)instruments), as well as other platforms still under development bycompanies such as Intelligent Biosystems and Pacific Biosystems.Although the chemistry by which sequence information is generated variesfor the different next-generation sequencing platforms, all of themshare the common feature of generating sequence data from a very largenumber of sequencing templates, on which the sequencing reactions arerun simultaneously. In general, the data from all of these sequencingreactions are collected using a scanner, and then assembled and analyzedusing computers and powerful bioinformatics programs. The sequencingreactions are performed, read, assembled, and analyzed in a “massivelyparallel” or “multiplex” fashion. The massively parallel nature of theseinstruments has required a change in thinking about what kind ofsequencing templates are needed and how to generate them in order toobtain the maximum possible amounts of sequencing data from thesepowerful instruments. Thus, rather than requiring genomic libraries ofDNA clones in E. coli, it is now necessary to think in terms of in vitrosystems for generating DNA fragment libraries comprising a collection orpopulation of DNA fragments generated from target DNA in a sample,wherein the combination of all of the DNA fragments in the collection orpopulation exhibits sequences that are qualitatively and/orquantitatively representative of the sequence of the target DNA fromwhich the DNA fragments were generated. In fact, in some cases, it isnecessary to think in terms of generating DNA fragment librariesconsisting of multiple genomic DNA fragment libraries, each of which islabeled with a different address tag or bar code to permitidentification of the source of each fragment sequenced.

In general, these next-generation sequencing methods requirefragmentation of genomic DNA or double-stranded cDNA (prepared from RNA)into smaller ssDNA fragments and addition of tags to at least one strandor preferably both strands of the ssDNA fragments. In some methods, thetags provide priming sites for DNA sequencing using a DNA polymerase. Insome methods, the tags also provide sites for capturing the fragmentsonto a surface, such as a bead (e.g., prior to emulsion PCRamplification for some of these methods; e.g., using methods asdescribed in U.S. Pat. No. 7,323,305). In most cases, the DNA fragmentlibraries used as templates for next-generation sequencing comprise 5′-and 3′-tagged DNA fragments or “di-tagged DNA fragments.” In general,current methods for generating DNA fragment libraries fornext-generation sequencing comprise fragmenting the target DNA that onedesires to sequence (e.g. target DNA comprising genomic DNA ordouble-stranded cDNA after reverse transcription of RNA) using asonicator, nebulizer, or a nuclease, and joining (e.g., by ligation)oligonucleotides consisting of adapters or tags to the 5′ and 3′ ends ofthe fragments.

There are a number of problems and inefficiencies with current methodsfor generating next-generation sequencing templates, as is illustratedby the workflow used at the Wellcome Trust Sanger Institute, one of theworld's largest genome centers (e.g., described in Quail, M A et al.,Nature Methods 5: 1005-1010, 2008). For example, Quail et al. found thatnebulization of genomic DNA for sequencing resulted in loss ofapproximately half of the DNA by mass and only about 5% of the originalDNA consisted of fragments in the approximately 200-bp size rangedesired for sequencing using the Illumina Genome Analyzer. They foundthat an alternative method, called “adapted focused acoustics” gavehigher yields of fragmented DNA and about 17% of the original DNAconsisted of fragments in the desired 200-bp size range, but even thisprocess is wasteful in terms of the sample or target DNA. Still further,the resulting DNA fragments often requires size selection by gelelectrophoresis, and additional steps to tag the size-selected DNAfragments, which is difficult, laborious, time-consuming, and expensive.

Thus, many of the methods currently used for fragmentation and taggingof double-stranded DNA for use in next-generation sequencing arewasteful of the DNA, require expensive instruments for fragmentation,and the procedures for fragmentation, tagging and recovering tagged DNAfragments are difficult, tedious, laborious, time-consuming,inefficient, costly, require relatively large amounts of sample nucleicacids. In addition, many of these methods generate tagged DNA fragmentsthat are not fully representative of the sequences contained in thesample nucleic acids from which they were generated. Thus, what isneeded in the art are methods for generating libraries of di-tagged DNAfragments in a massively parallel manner that overcome the limitationsof the current methods.

Some of the next-generation sequencing methods use circular ssDNAsubstrates in their sequencing process. For example, U.S. PatentApplication Nos. 20090011943; 20090005252; 20080318796; 20080234136;20080213771; 20070099208; and 20070072208 of Drmanac et al., eachincorporated herein by reference, discloses generation of circular ssDNAtemplates for massively parallel DNA sequencing. U.S. Patent ApplicationNo. 20080242560 of Gunderson and Steemers discloses methods comprising:making digital DNA balls (see, e.g., FIG. 8 in U.S. Patent ApplicationNo. 20080242560); and/or locus-specific cleavage and amplification ofDNA, such as genomic DNA, including for amplification by multipledisplacement amplification or whole genome amplification (e.g., FIG. 17therein) or by hyperbranched RCA (e.g., FIG. 18 therein) for generatingamplified nucleic acid arrays (e.g., ILLUMINA BeadArrays™; ILLUMINA, SanDiego Calif., USA).

What is needed are improved methods, compositions and kits for makingtagged circular ssDNA fragments from DNA from a biological sample (e.g.,from genomic DNA or mitochondrial DNA or episomal DNA, including DNAcloned in a plasmid, BAC, fosmid or other episomal vector) for use inamplification or DNA sequencing methods (such as the methods describedin U.S. Patent Application Nos. 2009/0011943; 2009/0005252;2008/0318796; 2008/0234136; 2008/0213771; 2007/0099208; and 2007/0072208of Drmanac et al.; or in U.S. Patent Application No. 2008/0242560 ofGunderson and Steemers or by Turner et al. of Pacific Biosciences.

Still further, some methods for amplification, such as whole genomeamplification, also require fragmentation and tagging of genomic DNA.Some of these methods are reviewed in: Whole Genome Amplification, ed.by S. Hughs and R. Lasken, 2005, Scion Publishing Ltd (on the worldwideweb at scionpublishing.com), incorporated herein by reference.

What is needed are improved methods for generating libraries of DNAfragments from target DNA molecules for amplification, includingamplification of whole or partial genomes from one organism (e.g, from aclinical sample) or from multiple organisms (e.g., metagenomic targetDNA from an environmental sample), for further analysis (e.g., byreal-time PCR, emulsion PCR, comparative genomic hybridization (CGH),comparative genomic sequencing (CGS), or for preparing DNA-specificlabeled probes (e.g., chromosome-specific probes, e.g., chromosomepaints, or e.g., gene-specific probes, e.g., for fluorescent in situhybridization (FISH), for a variety of purposes (e.g., for research,diagnostic, and industrial purposes).

Thus, what is needed in the art are better and more efficient methodsfor making libraries of tagged DNA fragments from target DNA for use innucleic acid analysis methods such as next-generation sequencing andamplification methods. What is needed are methods for generating DNAfragment libraries that do not require specialized instruments, and thatare easier, faster, require less hands-on time, can be performed withsmaller DNA samples and smaller volumes, are efficient in tagging one orboth ends of the fragments, and generate tagged DNA fragments that arequalitatively and quantitatively representative of the target nucleicacids in the sample from which they are generated.

SUMMARY OF THE INVENTION

The present invention relates to methods, compositions, and kits fortreating nucleic acid, and in particular, methods and compositions forfragmenting and tagging DNA using transposon compositions. The methods,compositions, and kits of the present invention are useful, for example,for generating libraries of tagged DNA fragments for use, e.g., in nextgeneration sequencing methods, fluorescence in situ hybridization, andthe like. In some preferred embodiments, the present invention relatesto preparation of linear ssDNA fragments or tagged circular ssDNAfragments (and amplification products thereof) from target DNAcomprising any dsDNA of interest (including double-stranded cDNAprepared from RNA), from any source, for genomic, subgenomic,transcriptomic, or metagenomic analysis, or analysis of RNA expression.

In some embodiments, the present invention provides methods forgenerating a library of tagged DNA fragments of a target DNA, comprisingincubating the target DNA with a transposase and a transposon end ortransposon end composition comprising a transferred strand that has atag domain in its 5′ portion, under conditions wherein a transpositionreaction is catalyzed by the transposase, and wherein the target DNA isfragmented to generate a plurality of target DNA fragments and atransferred strand of the transposon end or transposon end compositionis joined to the 5′ ends of each of a plurality of the target DNAfragments, to produce a plurality of 5′ tagged target DNA fragments. Insome embodiments, the methods further comprise incubating the pluralityof 5′-tagged target DNA fragments with at least one nucleic acidmodifying enzyme under conditions wherein a 3′ tag is joined to a 3′ endof the 5′-tagged target DNA fragment to produce a comprising di-taggedtarget DNA fragments.

In some embodiments, the present invention provides methods of tagging afragment of a target DNA, comprising incubating target DNA with atransposase and a transposon end or transposon end compositioncomprising a transferred strand comprising a tag domain in its 5′portion, under conditions wherein a transposition reaction is catalyzedby the transposase, wherein the target DNA is fragmented and thetransferred strand of the transposon end or transposon end compositionis joined to a 5′ end of a fragment of the target DNA to produce a5′-tagged target DNA fragment. In some preferred embodiments, themethods further comprise incubating the 5′ tagged target DNA fragmentwith a nucleic acid modifying enzyme under conditions wherein a 3′ tagis joined to a 3′ end of the 5′ tagged target DNA fragment to produce adi-tagged target DNA fragment. The methods are limited to the use of anyparticular nucleic acid modifying enzyme. For example, nucleic acidmodifying enzymes comprise polymerases, nucleases, ligases, and thelike. In some preferred embodiments, the nucleic acid modifying enzymecomprises a DNA polymerase, and the 3′ tag is formed by extension of the3′ end of the 5′ tagged target DNA fragment. In some embodiments the DNApolymerase comprises a template-dependent DNA polymerase, and in someembodiments, the DNA polymerase comprises a template independent DNApolymerase. In some preferred embodiments, the DNA polymerase is atemplate-dependent DNA polymerase that has strand-displacement and/or 5′nuclease activity.

In some embodiments, a nucleic acid modifying enzyme used in the presentmethods is a ligase, and the 3′ tag is formed by ligation of anoligonucleotide to the 3′ end of the 5′ tagged target DNA fragment. Insome embodiments the ligase comprises a template-dependent ligase, whilein some embodiments, the ligase comprises a template-independent ligase.

In some embodiments, the transferred ends comprise tag domains. Incertain preferred embodiments, the tag domains comprising one or more ofa restriction site domain, a capture tag domain, a sequencing tagdomain, an amplification tag domain, a detection tag domain, an addresstag domain, and a transcription promoter domain. In some embodiments,the tag domains are sequencing tag domains that comprise or consist ofsequencing tags selected from Roche 454A and 454B sequencing tags,ILLUMINA™ SOLEXA™ sequencing tags, Applied Biosystems' SOLID™ sequencingtags, the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polonysequencing tags, or the Complete Genomics sequencing tags.

Some embodiments further comprise amplifying one or more 5′ taggedtarget DNA fragments and/or di-tagged target DNA fragments. In somepreferred embodiments, the amplifying comprises use of one or more of aPCR amplification reaction, a strand-displacement amplificationreaction, a rolling circle amplification reaction, a ligase chainreaction, a transcription-mediated amplification reaction, or aloop-mediated amplification reaction. In certain preferred embodimentson the invention, amplifying comprises non-selectively amplifying 5′tagged target DNA fragments comprising a DNA fragment library ordi-tagged target DNA fragments comprising a DNA fragment library.

In some embodiments of the present invention the transposon endcomposition used on tagging a fragment or library comprises a pluralityof transferred strands that differ in nucleic acid sequence by at leastone nucleotide, and the amplifying comprises selectively amplifyingdi-tagged DNA fragments based on the nucleic acid sequences of the 5′end tags or tag domains. In other embodiments, the amplifying comprisesa polymerase chain reaction using a single oligonucleotide primer thatis complementary to the 3′ tag of the di-tagged target DNA fragments.

In some embodiments, the amplifying comprises a strand-displacementamplification reaction using a single oligonucleotide primer, in whichthe oligonucleotide primer consists of only ribonucleotides, or consistsof only purine ribonucleotides and only pyrimidine2′-F-2′-deoxyribonucleotides, and the strand displacement amplificationreaction comprises a strand-displacing DNA polymerase and a ribonucleaseH.

In some embodiments, the amplifying comprises a polymerase chainreaction using a first and a second oligonucleotide primer, eachcomprising 3′ end portions, wherein at least the 3′ end portion of thefirst PCR primer is complementary to the 3′ tag of the di-tagged targetDNA fragments, and wherein at least a the 3′-end portion of the secondPCR primer exhibits the sequence of at least a portion of the 5′ tag ortag domain of the di-tagged target DNA fragments. In certain preferredembodiments, the first or second oligonucleotide primer comprises a 5′end portion, wherein at least the 5′ end portion of the first primer isnot complementary to the 3′ tag of the di-tagged target DNA fragments,or wherein the 5′ portion of the second primer does not exhibit thesequence of at least a portion of the 5′ tag or tag domain of thedi-tagged target DNA fragments. In particularly preferred embodiments,the first and a second oligonucleotide primers each comprise 5′ endportions, wherein at least the 5′ end portion of the first PCR primer isnot complementary to the 3′ tag of the di-tagged target DNA fragments,and/or wherein the 5′-end portion of the second PCR primer does notexhibit the sequence of at least a portion of the 5′ tag domain of thedi-tagged target DNA fragments.

In some embodiments, the present invention provides methods ofgenerating a population of tagged circular single-stranded DNA fragmentsfrom a target DNA. In certain embodiments, this comprises incubating thetarget DNA with a transposase and a transposon end or a transposon endcomposition comprising a transferred strand that has a tag domain in its5′ portion and a transposon end in its 3′ portion, under conditionswherein a transposition reaction is catalyzed by the transposase, suchthat the target DNA is fragmented to generate a plurality of target DNAfragments and the transferred strand of the transposon end or transposonend composition is joined to the 5′ end of each of the plurality of thetarget DNA fragments to produce a population of 5′-tagged target DNAfragments. These methods further comprise steps of denaturing the5′-tagged target DNA fragments to produce single-stranded 5′-taggedtarget DNA fragments, and incubating the single-stranded 5′-taggedtarget DNA fragments with a nucleic acid ligase under conditions whereinthe single-stranded 5′-tagged target DNA fragments are intramolecularlyligated to form tagged circular single-stranded DNA fragments, eachexhibiting the sequences of the transferred strand and a portion of thetarget DNA.

In some embodiments, it is desirable to cleave such circularsingle-stranded DNA. Thus, in some embodiments of the methods of thepresent invention, the tag domain exhibits a sequence or structure of acleavage site, and the method further comprises: incubating the taggedcircular single-stranded DNA fragments with at least one enzymecomprising a cleavage enzyme composition wherein the cleavage enzymecomposition cleaves the tagged circular single-stranded DNA fragments toproduce di-tagged linear single-stranded DNA fragments. In certainpreferred embodiments, the cleavage enzyme composition comprises arestriction enzyme. In some embodiments, the tag domain exhibits asequence of a restriction site, and the method further comprisesannealing to the tagged circular single-stranded DNA fragmentsoligonucleotides complementary to the tag domain, and incubating thetagged circular single-stranded DNA fragments with a restrictionendonuclease that recognizes the restriction site, wherein therestriction endonuclease cleaves the tagged circular single-stranded DNAfragments to produce di-tagged linear single-stranded DNA fragments.

In some embodiments, it is useful to amplify the fragments and librariesof the invention. Thus, some embodiments, further comprise amplifyingone or more of the tagged circular single-stranded DNA fragments and/orthe di-tagged linear single-stranded DNA fragments. In certain preferredembodiments, the amplifying comprises a polymerase chain reaction usinga first and a second oligonucleotide primer, each comprising 3′ endportions, wherein at least the 3′ end portion of the first PCR primer iscomplementary to at least a portion of the sequence of the transferredstrand in the tagged circular single-stranded DNA fragments or in thedi-tagged linear single-stranded DNA fragments, and wherein at least athe 3′-end portion of the second PCR primer is complementary at least aportion of the complement of the transferred strand in the taggedcircular single-stranded DNA fragments or in the di-tagged linearsingle-stranded DNA fragments.

In some embodiments in which tagged circular single-stranded DNAfragments or the di-tagged linear single-stranded DNA fragments areamplified, the first and second oligonucleotide primers each comprise 5′end portions, wherein the 5′ end portion of the first PCR primer is notcomplementary to the sequence of the transferred strand in the taggedcircular single-stranded DNA fragments or in the di-tagged linearsingle-stranded DNA fragments, and wherein the 5′-end portion of thesecond PCR primer is not complementary the complement of the transferredstrand in the tagged circular single-stranded DNA fragments or in thedi-tagged linear single-stranded DNA fragments.

In some embodiments, the present invention provides methods ofgenerating a population of tagged circular DNA fragments from a targetDNA, comprising incubating target DNA with a transposase and a hairpintransposon end composition comprising an oligonucleotide that exhibits anon-transferred strand sequence at its 5′ end, a transferred strandsequence at its 3′ end, and an intervening loop sequence comprising atag domain, under conditions wherein the oligonucleotide can form anintramolecular stem-loop, and wherein a transposition reaction iscatalyzed by the transposase, such that the target DNA is fragmented togenerate a plurality of target DNA fragments, and the oligonucleotide ofthe hairpin transposon end composition is joined to the 5′ end of eachof the plurality of target DNA fragments to produce a population of5′-tagged target DNA fragments. In some embodiments, the method furthercomprises filling gaps and ligating nicks in the fragment molecules. Insome embodiments, this comprises incubating the population of 5′-taggedtarget DNA fragments with a template-dependent ligase and a DNApolymerase that lacks 5′ to 3′ exonuclease, 3′ to 5′ exonuclease, andstrand-displacement activities, or one or more sizes of random-sequenceoligonucleotides, which, alone, or in combination, have the same lengthas the single-stranded gaps in the 5′-tagged DNA fragments that resultfollowing a transposition reaction with the transposase and the hairpintransposon end composition, under conditions wherein single-strandedgaps in the 5′-tagged target DNA fragments are filled and the 3′ end ofeach 5′-tagged DNA fragment is joined to the 5′-end of another 5′-taggedDNA fragment that comprises a complementary portion of the target DNA,to form tagged circular DNA fragments comprising the tag domain in loopstructures and both strands of a portion of the target DNA. In certainpreferred embodiments, the filling and joining comprises incubating the5′ tagged DNA fragments from with the DNA polymerase under conditionswherein the 3′ end of each 5′-tagged DNA fragment is extended to form apopulation of 5′-tagged DNA fragment extension products, and incubatingthe 5′-tagged DNA fragment extension products with thetemplate-dependent ligase under conditions wherein the 5′-tagged DNAfragment extension products are ligated, thereby generating the taggedcircular DNA fragments. In particularly preferred embodiments, the DNApolymerase and the ligase are provided in a mixture, and the filling andligating are carried out in a single reaction mixture.

In some embodiments, the filling and ligating steps comprise incubatingthe 5′ tagged DNA fragments with the one or more sizes ofrandom-sequence oligonucleotides and the template-dependent ligase underconditions wherein the random-sequence oligonucleotides anneal and fillsingle-stranded gaps and are ligated to each other or to adjacent endsof 5′ tagged DNA fragments to form tagged circular DNA fragments.

In preferred embodiments, the method further comprises separating thetagged circular DNA fragments from linear DNA, unligated random sequenceoligonucleotides, and/or hairpin transposon end composition not joinedto target DNA. In particularly preferred embodiments, the reactionmixture containing the tagged circular DNA fragments is treated with T5exonuclease to remove linear DNA, such as unligated fragments andrandom-sequence oligonucleotides.

In some embodiments, it is desirable to cleave the tagged circular DNAmolecules. For example, in some embodiments, the method furthercomprises a step of: cleaving the tagged circular DNA fragments in theloop structures to generate fantail double-stranded DNA fragments, eachstrand of which has a portion of the tag on its 5′-end and a portion ofthe tag on its 3′-end. Thus, in some embodiments of the methods of thepresent invention, the tag domain in the loop structures exhibit asequence or structure of a cleavage site, and the method furthercomprises: incubating the tagged circular single-stranded DNA fragmentswith at least one enzyme comprising a cleavage enzyme compositionwherein the cleavage enzyme composition cleaves the tagged circularsingle-stranded DNA fragments to produce fantail double-stranded DNAfragments.

In some embodiments, the cleavage enzyme composition comprises anN-glycosylase and an AP endonuclease. In certain preferred embodiments,the cleavage enzyme is an N-glycosylase selected from amonguracil-N-glycosylase and an AP endonuclease and FPG protein and the APendonuclease is selected from among E. coli endonuclease III orendonuclease IV.

In certain preferred embodiments, the cleavage enzyme compositioncomprises a restriction enzyme. In some embodiments, the tag domainexhibits a sequence of a restriction site, and the method furthercomprises annealing to the tagged circular single-stranded DNA fragmentsoligonucleotides complementary to the tag domain, and incubating thetagged circular single-stranded DNA fragments with a restrictionendonuclease that recognizes the restriction site, wherein therestriction endonuclease cleaves the tagged circular single-stranded DNAfragments to produce fantail double-stranded DNA fragments, each strandof which has a portion of the tag on its 5′-end and a portion of the tagon its 3′-end.

Some embodiments comprise additionally comprising denaturing the fantaildouble-stranded DNA fragments to generate di-tagged linearsingle-stranded DNA fragments.

The circular and fantail embodiments fined use in methods comprisingusing the DNA fragments as templates in a DNA sequencing method or anamplification reaction. Thus, in some embodiments, the methods of thepresent invention further comprise amplifying tagged circular DNAfragments, fantail double-stranded DNA fragments and/or di-tagged linearsingle-stranded DNA fragments. In preferred embodiments, amplifyingcomprises use of one or more of a PCR amplification reaction, astrand-displacement amplification reaction, a rolling circleamplification reaction, a ligase chain reaction, atranscription-mediated amplification reaction, or a loop-mediatedamplification reaction. In particularly preferred embodiments, theamplifying comprises a polymerase chain reaction using a first and asecond oligonucleotide primer, each comprising 3′ end portions, whereinat least the 3′ end portion of the first PCR primer is complementary toat least a portion of the tag domain, and wherein at least a the 3′-endportion of the second PCR primer exhibits the sequence of at least aportion of the tag domain. In some embodiments, the first and secondoligonucleotide primers each comprise 5′ end portions, wherein the 5′end portion of the first PCR primer is not complementary to the tagsequence, and wherein the 5′-end portion of the second PCR primer doesnot exhibit the sequence of the tag domain.

Preferred embodiments of any of the PCR amplification described abovecomprise amplifications wherein the 5′ end portions of the first and/orthe second PCR primers exhibit tag domains. In still more preferredembodiments, the tag domains comprise one or more of a restriction sitedomain, a capture tag domain, a sequencing tag domain, an amplificationtag domain, a detection tag domain, an address tag domain, and atranscription promoter domain.

In particularly preferred embodiments of the methods described herein,the tag domains are sequencing tag domains that comprise or consist ofsequencing tags selected from Roche 454A and 454B sequencing tags,ILLUMINA™ SOLEXA™ sequencing tags, Applied Biosystems' SOLID™ sequencingtags, the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polonysequencing tags, or the Complete Genomics sequencing tags.

In some embodiments, the present invention provides methods of tagging afragment of a target DNA, comprising incubating target DNA with atransposase and a transposon end composition comprising a transferredstrand that exhibits, in its 5′ portion, the sequence of a tag domainthat is not a transposon end, and, in its 3′ portion, the sequence ofthe transferred transposon end, wherein the target DNA is fragmented,and the transferred strand of the transposon end composition is joinedto a 5′ end of a fragment of the target DNA to produce a 5′ taggedtarget DNA fragment.

In some embodiments, the present invention provides methods of producinga 5′ tagged DNA fragment library, comprising incubating the target DNAwith a transposase and a transposon end composition comprisingtransferred strands that exhibit, in their 5′ portions, the sequence ofone or more tag domains for a particular purpose, and, in their 3′portions, the sequence of the transferred transposon end, underconditions wherein a transposition reaction is catalyzed by thetransposase, wherein the target DNA is fragmented, and the transferredstrands of the transposon end composition are joined to 5′ ends of afragments of the target DNA to produce 5′-tagged target DNA fragments,such that the transposition reaction produces a plurality of 5′-taggedtarget DNA fragments comprising a DNA fragment library from the targetDNA.

The present invention also provides compositions. For example, in someembodiments, the present invention provides a composition comprising asynthetic nucleic acid molecule having a 5′ portion comprising a tagdomain and a 3′ portion comprising a transferred strand of a transposonend. In some embodiments, the invention provides a compositioncomprising a plurality of synthetic nucleic acid molecules, wherein thenucleic acid molecules comprise 5′ portions comprising tag domains thatdiffer by at least one nucleotide, and 3′ portions comprising atransferred strand of a transposon end.

In some embodiments of the compositions described above, at least the 3′portion of the nucleic acid molecule is double-stranded DNA. In certainpreferred embodiments, the transposon end is a Tn5 transposon end, whilein other embodiments, the transposon end is a Mu transposon end.

In some embodiments, the tag domain of the nucleic acid moleculecomposition comprises one or more of a restriction site domain, acapture tag domain, a sequencing tag domain, an amplification tagdomain, a detection tag domain, an address tag domain, and atranscription promoter domain. In particularly preferred embodiments,the tag domains comprise a sequencing tags comprising or consisting ofsequencing tags selected from Roche 454A and 454B sequencing tags,ILLUMINA™ SOLEXA™ sequencing tags, Applied Biosystems' SOLID™ sequencingtags, the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polonysequencing tags, or the Complete Genomics sequencing tags.

In some embodiments, the composition comprising a nucleic acid moleculefurther comprises a purified transposase. In preferred embodiments, thetransposase is selected from a Tn5 transposase and a Mu transposase. Inpreferred embodiments, the nucleic acid molecule and the transposase areprovided in a mixture. In particularly preferred embodiments, themixture further comprises a nonionic detergent. In still more preferredembodiments, the non-ionic detergent comprises Nonidet P-40 and/orTween-20.

In some embodiments, the present invention provides a kit comprising anyof the compositions described above or elsewhere herein. In someembodiments, the kit further comprises one or more of a ligase, apolymerase, and/or reagents for an amplification reaction. Inparticularly preferred embodiments, the reagents for an amplificationreaction comprise reagents for a polymerase chain reaction. In preferredembodiments, the reagents or an amplification reaction and/or polymerasechain reaction comprise at least one primer, and in particularlypreferred embodiments, the reagents comprise a primer wherein comprisinga 3′ portion that is complementary to the complement of the tag domainof the 5′ portion of the nucleic acid molecule. In preferredembodiments, the tag domain comprises one or more of a restriction sitedomain, a capture tag domain, a sequencing tag domain, an amplificationtag domain, a detection tag domain, an address tag domain, and atranscription promoter domain, and in particularly preferredembodiments, the tag domain comprises a sequencing tag domain thatcomprises or consists of a sequencing tag selected from Roche 454A and454B sequencing tags, ILLUMINA™ SOLEXA™ sequencing tags, AppliedBiosystems' SOLID™ sequencing tags, the Pacific Biosciences' SMRT™sequencing tags, Pollonator Polony sequencing tags, or the CompleteGenomics sequencing tags.

In some embodiments, a kit of the present invention further comprisesreagents for a DNA sequencing reaction.

In some embodiments, the present invention comprises a reaction mixturecomprising a double-stranded target DNA and any of the tagged transposonend nucleic acid molecules and/or transposase compositions describedabove.

In some embodiments, the present invention provides a compositioncomprising a purified transposase and a plurality of synthetictransposon ends or transposon end compositions. In some embodiments, thetransposon end compositions comprise hairpin transposon ends, while insome embodiments, the synthetic transposon ends or transposon endcompositions comprise separate transferred strands and non-transferredstrands. In some preferred embodiments, the transferred strands comprise5′ tag domains, e.g., comprising one or more of a restriction sitedomain, a capture tag domain, a sequencing tag domain, an amplificationtag domain, a detection tag domain, an address tag domain, and atranscription promoter domain. In particularly preferred embodiments,the tag domains comprise sequencing tags comprising or consisting ofsequencing a tag selected from Roche 454A and 454B sequencing tags,ILLUMINA™ SOLEXA™ sequencing tags, Applied Biosystems' SOLID™ sequencingtags, the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polonysequencing tags, or the Complete Genomics sequencing tags.

In some embodiments, the composition comprising a purified transposasecomprises a plurality of synthetic transposon ends comprise at least twotransferred strands that differ from each other by at least onenucleotide, and in preferred embodiments, the transferred strandscomprise 5′ portions and 3′ portions, wherein at least two of the 5′portions of the transferred strands comprise tags that differ from eachother by at least one nucleotide, and wherein the 3′ portions of thetransferred strands comprise a the same transposon end sequence.

In some embodiments the transposon ends comprise Mu transposon ends andthe transposase is Mu transposase, and in some preferred embodiments,the 3′ portions of the transferred strands comprise a sequence from a Mutransposon end, and wherein the 5′ portions of the transferred strandsare not from a Mu transposon.

In some embodiments the transposon ends comprise Tn5 transposon ends andthe transposase is Tn5 transposase, and in some preferred embodiments,the 3′ portions of the transferred strands comprise a sequence from aTn5 transposon end, and wherein the 5′ portions of the transferredstrands are not from a Tn5 transposon.

In some embodiments, the present invention provides compositionscomprising a DNA fragment library, wherein the DNA fragment librarycomprises fragments of the target DNA having 5′ ends comprisingsequences from transferred strands from transposon ends or transposonend compositions. In preferred embodiments, the sequences from thetransferred strands comprise 5′ tag domains and in still more preferredembodiments, the DNA fragment library comprises fragments of target DNAcomprising 3′ tags complementary to a transferred strand from atransposon end or transposon end composition. In some embodiments, theDNA fragment library comprises double-stranded fragments of the targetDNA.

In some embodiments, the present invention provides compositionscomprising a tagged circular DNA fragment of a target DNA, comprising aportion comprising non-transferred strand sequence at the 5′ end of theportion, a transferred strand sequence at the 3′ end or the portion, anintervening loop sequence comprising a tag domain, sequences of bothstrands of a portion of a target DNA.

In some embodiments, the present invention provides compositionscomprising a tagged circular single-stranded DNA fragment of a targetDNA, comprising transferred strand sequence from a transposon end or atransposon end composition, and a single-stranded portion of a targetDNA. In preferred embodiments, the transferred strand sequence comprisesa tag domain.

In some embodiments, the present invention provides compositionscomprising a fantail double-stranded DNA fragment of a target DNA,comprising a double-stranded portion of a target DNA wherein each strandhas a 5′ end comprising at least a portion of a transferred strandsequence, and a 3′ end comprising at least a portion of anon-transferred strand sequence.

Embodiments of the invention are described in this summary, and in theDetailed Description of the Invention, below, which is incorporated hereby reference. Although the invention has been described in connectionwith specific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 provides a schematic diagram showing insertion of a transposoninto a target DNA in a transposition reaction.

FIG. 2 provides a schematic diagram showing fragmentation and tagging oftarget DNA by insertion of transposon ends in a transposition reaction.

FIG. 3 provides a schematic diagram of the products of twotransposase-catalyzed transposon end composition insertion events. Thus,the product of the transposase-catalyzed transposon end compositioninsertion depicted in the left of the figure shows a transposon endorientation wherein the transferred strand of the transposon endcomposition (i.e., wherein the transferred transposon end exhibits thesequence 5′ AGATGTGTATAAGAGACAG 3′ (SEQ ID NO:1), with its 3′-end joinedto the target DNA) is in the top strand, and the product of thetransposase-catalyzed transposon end composition insertion depicted inthe right of the figure shows a transposon end orientation wherein thetransferred strand is in the bottom strand. The non-transferred strandis SEQ ID NO:2.

FIG. 4 illustrates examples of two different tagged transposon ends,each comprising a transferred strand oligonucleotide with a differenttag in the 5′-portion for use in generating a library of tagged DNAfragments. Extending the 3′ ends of each strand using, e.g., DNApolymerase having 5′ nuclease or strand-displacement activity, producesdi-tagged ssDNA fragments. The transferred strand sequence shown is SEQID NO:1; the non-transferred strand is SEQ ID NO:2.

FIG. 5 shows an image of agarose gel showing the size range of 5′-taggedDNA fragment transposition products produced using different transposomeconcentrations.

FIG. 6 shows an image of agarose gel showing the size range of 5′-taggedDNA fragment transposition products produced in five minute reactions atdifferent temperatures, with different reaction buffers, in the presenceor absence of dimethylformamide (DMF).

FIG. 7 illustrates an example of the method wherein a DNA polymerasethat has strand-displacement DNA polymerase activity and/or that has5′-to-3′ exonuclease activity is used to join the complement of thetransferred strand to the 5′-tagged DNA fragments from the in vitrotransposition reaction to generate a library of DNA fragments comprisingdi-tagged ssDNA fragments. As shown, the strand-displacement and/or5′-to-3′ exonuclease activity of the DNA polymerase displaces or digeststhe DNA that is annealed downstream of the DNA polymerase extensionproduct and the extension by the DNA polymerase joins a second tag thatcomprises or consists of a DNA sequence that is complementary to thefirst tag inserted into the opposite strand. In some embodiments, thedi-tagged DNA fragment products are PCR amplified using oligonucleotidesthat are complementary to the complement of the transferred strand. Thetransferred strand sequence shown is SEQ ID NO:1; the non-transferredstrand is SEQ ID NO:2.

FIG. 8 illustrates an example of the method wherein a DNA polymerasethat has strand-displacement DNA polymerase activity and/or that has5′-to-3′ exonuclease activity is used to join the second tag to the5′-tagged DNA fragments from the in vitro transposition reaction togenerate a library of DNA fragments comprising di-tagged DNA fragments.As shown, the strand-displacement and/or 5′-to-3′ exonuclease activityof the DNA polymerase displaces or digests the DNA that is annealeddownstream of the DNA polymerase extension product and the extension bythe DNA polymerase joins a second tag that comprises or consists of aDNA sequence that is complementary to the first tag inserted into theopposite strand. In some embodiments, the di-tagged ssDNA fragmentproducts are PCR amplified using oligonucleotides that are complementaryto the different sequences in the respective first or second tags as PCRprimers. The transferred strand sequence shown is SEQ ID NO:1; thenon-transferred strand is SEQ ID NO:2.

FIG. 9 provides a comparison of sequencing read length, accuracy, andcoverage of a single contig using a DNA fragment library producedaccording to embodiments of the present invention, compared to a controllibrary produced using nebulization.

FIG. 10 provides a schematic diagram showing fragmentation and taggingof target DNA to produce a Roche/454-compatible library by insertion oftagged transposon ends in a transposition reaction, followed bytag-specific PCR+/−barcodes.

FIG. 11 shows an image of agarose gel showing input DNA (lane 2), thesize range of 5′-tagged DNA fragment transposition products (lane 3),the size range of PCR reaction products (lane 4), and a control reaction(lane 5) for the preparation of a bar coded Roche/454 FLX-compatiblesequencing library as illustrated in FIG. 10.

FIG. 12 shows an image of an agarose gel showing the size range of5′-tagged DNA fragment transposition products (lane 2), the size rangeof PCR reaction products (lane 3), and a control reaction (lane 5) forthe preparation of a Roche/454 FLX Titanium-compatible sequencinglibrary from amplicon DNA, similar to the method illustrated in FIG. 10.

FIG. 13 provides a schematic diagram showing fragmentation and taggingof target DNA to produce an Illumina/Solexa compatible library byinsertion of tagged transposon ends in a transposition reaction,followed by tag-specific PCR+/−barcodes.

FIG. 14 shows an image of agarose gel showing input DNA (lane 2), thesize range of 5′-tagged DNA fragment transposition products (lane 3),the size range of PCR reaction products (lane 4), and a control reaction(lane 5) for the preparation of a bar coded Illumina GAII-compatiblesequencing library as illustrated in FIG. 13.

FIG. 15 compares the process and complexity of prior art methods oflibrary preparation with DNA fragment library preparation according toembodiments of the present invention.

FIG. 16. shows an example of a product of the method of the inventionfollowing incubation of a transposase (e.g., EZ-Tn5® Transposase here)and a hairpin transposon end composition (e.g., the EZ-Tn5™“pMETS-N-MENTS” hairpin transposon end composition, SEQ ID NO:3,depicted here) in an in vitro transposase reaction in the presence ofdouble-stranded target DNA (e.g., genomic DNA or double-stranded cDNA).

FIG. 17. Fragmentation and Tagging of Target DNA with a HairpinTransposon End Composition. FIG. 17A shows a schematic diagram of aproduct (among a population of many such products) resulting from twotransposase-catalyzed insertion events of the hairpin transposon endcomposition into target DNA. Briefly, target DNA (e.g., comprisingdouble-stranded genomic DNA or cDNA) is incubated in an in vitrotransposase reaction containing a transposase and a hairpin transposonend composition (e.g., EZ-Tn5™ Transposase and an EZ-Tn5™ hairpintransposon end composition). The transferred end sequence of eachinserted hairpin transposon end composition is joined via a loopstructure to the non-transferred-strand sequence of the transposon end.The loop can have any arbitrary tag domain, such as a restriction sitedomain, a capture tag domain, a sequencing tag domain, a detection tagdomain, an address tag domain, a transcription promoter domain, or anamplification tag domain. For example, the sequencing tag domain canexhibit the sequence of a Roche 454A or 454B sequencing tag. Forexample, in this figure, the sequencing tag domain exhibits one or moresequences in the loop between the complementary transposon-end sequences(the stem). FIG. 17B shows an SYBR Gold-stained 1% agaroseelectrophoresis gel of the products of 5′-tagging and fragmentation of 1μg of T7 D111 genomic dsDNA using 0, 0.5, 1, 2, or 3 μM of thepMETS-N-MENTS hairpin transposon end composition and equimolar amountsof EZ-Tn5™ transposase (EPICENTRE Biotechnologies, Madison, Wis.), afterincubation in 33 mM Tris-acetate pH 7.6, 66 mM KOAc, and 10 mM Mg(OAc)₂for 2 hours at 37° C.

FIG. 18. Generation of 5′-Tagged Circular DNA Templates. FIG. 18A is aschematic diagram of one embodiment of the method. Briefly, 9-nucleotidegaps generated by two insertions of the hairpin transposon endcomposition into a target DNA are filled by extension of the 3′ ends ofthe 5′-tagged DNA fragments generated using a DNA polymerase that lacks5′-to-3′ exonuclease and strand-displacement activities (e.g., T4 DNApolymerase) and the single-stranded DNA in the gap regions as templates,and then the DNA polymerase extension products of the 5′-tagged DNAfragments are ligated using a template-dependent ligase (e.g., E. coliDNA ligase) to generate tagged circular DNA fragments. The taggedcircular DNA fragments are resistant to T5 exonuclease, which is used inthe embodiment in the figure to remove unligated linear single-strandedand double-stranded DNA. FIG. 18B shows a SYBR Gold-stained 1% agaroseelectrophoresis gel of the reaction products generated in the presenceor absence of T4 DNA polymerase and/or E. coli DNA ligase. As shown,tagged circular DNA fragments, which are resistant to treatment by T5exonuclease, were generated only in the presence of both the T4 DNApolymerase and the E. coli DNA ligase.

FIG. 19 illustrates use of a terminal transferase to join a second (3′)tag to 5′-tagged DNA fragments, to generate a library of DNA fragmentscomprising 5′- and 3′-tagged (“di-tagged”) ssDNA fragments. In theillustrated embodiment, a sequencing tag domain comprising a sequencingtag (SEQ) is added using a template-dependent DNA polymerase.

FIG. 20 illustrates use of a terminal tagging oligonucleotide as atemplate to add a second (3′) tag to 5′-tagged ssDNA fragments togenerate a library of DNA fragments comprising di-tagged ssDNAfragments.

FIG. 21 illustrates an embodiment wherein 5′-tagged DNA fragments areincubated in the presence of a DNA ligase and a ligation taggingoligonucleotide that comprises a 5′ portion that has a phosphate groupon its 5′-end and that exhibits a random sequence that anneals to the9-base gap or region of single-stranded DNA that results from the EZ-Tn5transposase-catalyzed insertion of EZ-Tn5 ME transposon end into thetarget DNA, and a 3′ portion that exhibits a second tag sequence (tag#2). In this example, the ligation tagging oligonucleotide has a5′-portion that exhibits a 6-nucleotide random sequence. The randomsequence anneals to the single-stranded annealed target DNA in the9-base gap regions that result from insertion of the transposon ends(e.g. here, the 19-basepair EZ-Tn5 mosaic end, SEQ ID NO:1 and SEQ IDNO:2 or ME transposon end) into the double-stranded target DNA. Thoseligation tagging oligonucleotides that anneal to the single-strandedtarget DNA in the gap regions so that a 5′-phosphorylated end abuts the3′-end of a 5′-tagged DNA fragment are then joined by the nucleic acidligase in a template-dependent ligation reaction, thereby generating 5′-and 3′-tagged DNA fragments with the first tag on the 5′ end and thesecond tag on the 3′-end. Thus, if transposon end insertions occur intoboth strands of the target DNA in close proximity (e.g., sites in thetarget DNA that are within about 50 Kb, about 40 Kb, about 30 Kb, about20 Kb, about 10 Kb, about 5 Kb, about 1 Kb, about 500 bp, or preferably,within about 150 by to about 500 by of each other), the dual-taggedstrands can be purified, PCR-amplified, optionally, labeled with adetectable dye (e.g., for use as target for annealing to a microarray,e.g., for expression analysis), or, first captured on a surface (e.g.,on a bead; e.g. a bead for next-generation sequencing) and thenamplified (e.g., using emulsion PCR, e.g., for use as next-generationsequencing templates; or using limited-cycle PCR, e.g., for use incopy-number variation (CNV) experiments, e.g., by comparative genomichybridization on a microarray).

FIG. 22 (A) shows a schematic diagram of an embodiment of DNAfragmentation, tagging and circularization of genomic DNA using themethod of the invention. The pointed box with black line represents thep454.1MEDS transposon end composition. Dashed lines represent T7 D111genomic dsDNA fragments.

FIG. 22 (B) shows an agarose gel of the products of 5′-tagging andfragmentation of T7 D111 genomic dsDNA using p454.1MEDS transposon endcomposition and EZ-Tn5™ transposase (EPICENTRE Biotechnologies). One μgof T7 D111 genomic dsDNA was incubated with or without 0.5 μM of thep454.1MEDS transposon end composition in 33 mM Tris-acetate pH 7.6, 66mM KOAc, and 10 mM Mg(OAc)₂ for 1 hour at 37° C., either in the presenceor the absence of EZ-Tn5 transposase as indicated. The 5′-tagged linearssDNA reaction products were resolved by electrophoresis in a 1% agarosegel and stained with SYBR Gold.

FIG. 23 (A) shows a schematic of PCR amplification of the taggedcircular ssDNA fragments using the pMETS and the p454.1 oligonucleotidesas PCR primers.

FIG. 23 (B) shows an agarose gel of the PCR amplification productsobtained when tagged circular ssDNA fragments obtained using the methodof the invention were amplified by PCR using the pMETS and pc454.1oligonucleotides as PCR primers. First, the 5′-tagged fragmented dsDNA,obtained as shown in FIG. 22 (B), was denatured by heating 95° C. for 3minutes and rapidly cooling on ice. A portion of the resulting denatured5′-tagged linear ssDNA fragments were incubated in 33 mM Tris-acetate pH7.6, 66 mM KOAc, 2.5 mM MnCl₂, and 1M betaine for 2 hours at 60° C. inthe presence or the absence of 400 units of CIRCLIGASE™ ssDNA ligase asdescribed in Example 17 in order to circularize the 5′-tagged linearssDNA fragments. Reaction products were incubated with exonuclease I andexonuclease III for 1 hour at 37° C. to digest linear ssDNA fragmentsprior to PCR amplification. The tagged circular ssDNA fragments werethen amplified by PCR using the pMETS and pc454.1 oligonucleotides asPCR primers. PCR products were resolved by 1% agarose electrophoresisand visualized by SYBR Gold staining.

DEFINITIONS

Unless specifically defined or described differently elsewhere herein,the following terms and descriptions related to the invention shall beunderstood as given below.

When the terms “for example”, “e.g.”, “such as”, “include”, “including”or variations thereof are used herein, these terms will not be deemed tobe terms of limitation, and will be interpreted to mean “but not limitedto” or “without limitation.”

The use of terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

As used herein, the terms “isolated,” “to isolate,” “isolation,”“purified,” “to purify,” “purification,” and grammatical equivalentsthereof as used herein, unless specified otherwise, refer to thereduction in the amount of at least one contaminant (such as proteinand/or nucleic acid sequence) from a sample or from a source (e.g., acell) from which the material is isolated. Thus purification results inan “enrichment,” i.e., an increase in the amount of a desirable proteinand/or nucleic acid sequence in the sample.

As used herein, a “tag” refers to a non-target nucleic acid component,generally DNA, that provides a means of addressing a nucleic acidfragment to which it is joined. For example, in preferred embodiments, atag comprises a nucleotide sequence that permits identification,recognition, and/or molecular or biochemical manipulation of the DNA towhich the tag is attached (e.g., by providing a site for annealing anoligonucleotide, such as a primer for extension by a DNA polymerase, oran oligonucleotide for capture or for a ligation reaction). The processof joining the tag to the DNA molecule is sometimes referred to hereinas “tagging” and DNA that undergoes tagging or that contains a tag isreferred to as “tagged” (e.g., “tagged DNA”).”

As used herein, the term “ligase” refers to a nucleic acid modifyingenzyme that catalyzes intra- and intermolecular formation ofphosphodiester bonds between 5′-phosphate and 3′-hydroxyl termini ofnucleic acid strands. Ligases include, e.g., template-independentligases, such as CIRCLIGASE™ ssDNA ligase, that can join ends ofsingle-stranded RNA and DNA, and template-dependent or homologousligases, that seal nicks in double-stranded DNA (example describedbelow).

As used herein, a “homologous ligase” or “template-dependent ligase”means a DNA ligase that catalyzes intra- and intermolecular formation ofphosphodiester bonds between 5′-phosphate and 3′-hydroxyl termini of DNAstrands that are adjacent to each other when annealed to a complementarypolynucleotide. Some embodiments of intramolecular ligation produce acircular molecule and are referred to as “circularization”. Thepolynucleotide to which both ends of the DNA ends to be ligated annealadjacently is referred to herein as a “ligation template” and theligation is referred to as “homologous ligation” or “template-dependentligation.” The ligation template can be a complementary DNA sequence ingenomic or other DNA in a biological sample (in which case, it is oftenreferred to as a “target sequence”), or the ligation template can be a“bridging oligodeoxyribonucleotide” or “ligation splintoligodeoxyribonucleotide” (or “ligation splint”) that is synthesizedand/or provided specifically for use in a particular assay or method.Examples of homologous or template-dependent DNA ligases includeNAD-type DNA ligases such as E. coli DNA ligase, Tth DNA ligase, Tfl DNAligase, and AMPLIGASE® DNA ligase (EPICENTRE Biotechnologies, Madison,Wis., USA), which catalyze intramolecular ligation of ssDNA moleculesonly in the presence of a ligation template, and ATP-type DNA ligases,such as T4 DNA ligase or FASTLINKT™ DNA ligase (EPICENTREBiotechnologies), which, while they do not require a ligation templatefor blunt-end ligation, they catalyze template-dependent ligation muchmore efficiently.

In some preferred embodiments, the template-dependent ligase is from apsychrophilic bacterium or a psychrophilic bacteriophage so that theligation can be performed at lower temperatures (e.g., when thesequences of the oligonucleotides or polynucleotides that form theligation junction exhibit lower T_(m)'s). A DNA ligase is chosen for usein the method that is active at a temperature at which the DNA moleculesused for joining (e.g., the 5′-tagged DNA fragment extension products orthe 5′-tagged DNA fragments and the random-sequence oligonucleotides)anneal for sufficient time to be ligated by the ligase.

An important step in embodiments of the method of the present inventionis the use of an in vitro transposition reaction to fragment and tag thetarget DNA to generate tagged DNA fragments. The in vitro transpositionreaction requires a transposase, a transposon end composition, andsuitable reaction conditions.

A “transposase” means an enzyme that is capable of forming a functionalcomplex with a transposon end-containing composition (e.g., transposons,transposon ends, transposon end compositions) and catalyzing insertionor transposition of the transposon end-containing composition into thedouble-stranded target DNA with which it is incubated in an in vitrotransposition reaction.

The term “transposon end” means a double-stranded DNA that exhibits onlythe nucleotide sequences (the “transposon end sequences”) that arenecessary to form the complex with the transposase or integrase enzymethat is functional in an in vitro transposition reaction. A transposonend forms a “complex” or a “synaptic complex” or a “transposome complex”or a “transposome composition with a transposase or integrase thatrecognizes and binds to the transposon end, and which complex is capableof inserting or transposing the transposon end into target DNA withwhich it is incubated in an in vitro transposition reaction. Atransposon end exhibits two complementary sequences consisting of a“transferred transposon end sequence” or “transferred strand” and a“non-transferred transposon end sequence,” or “non transferred strand”For example, one transposon end that forms a complex with a hyperactiveTn5 transposase (e.g., EZ-Tn5™ Transposase, EPICENTRE Biotechnologies,Madison, Wis., USA) that is active in an in vitro transposition reactioncomprises a transferred strand that exhibits a “transferred transposonend sequence” as follows:

5′ AGATGTGTATAAGAGACAG 3′, (SEQ ID NO: 1)

and a non-transferred strand that exhibits a “non-transferred transposonend sequence” as follows:

5′ CTGTCT CTTATACACATCT 3′. (SEQ ID NO: 2)

The 3′-end of a transferred strand is joined or transferred to targetDNA in an in vitro transposition reaction. The non-transferred strand,which exhibits a transposon end sequence that is complementary to thetransferred transposon end sequence, is not joined or transferred to thetarget DNA in an in vitro transposition reaction.

In some embodiments, the transferred strand and non-transferred strandare covalently joined. For example, in some embodiments, the transferredand non-transferred strand sequences are provided on a singleoligonucleotide, e.g., in a hairpin configuration. As such, although thefree end of the non-transferred strand is not joined to the target DNAdirectly by the transposition reaction, the non-transferred strandbecomes attached to the DNA fragment indirectly, because thenon-transferred strand is linked to the transferred strand by the loopof the hairpin structure.

A “transposon end composition” means a composition comprising atransposon end (i.e., the minimum double-stranded DNA segment that iscapable of acting with a transposase to undergo a transpositionreaction), optionally plus additional sequence or sequences. 5′-of thetransferred transposon end sequence and/or 3′-of the non-transferredtransposon end sequence. For example, a transposon end attached to a tagis a “transposon end composition.” In some embodiments, the transposonend composition comprises or consists of two transposon endoligonucleotides consisting of the “transferred transposon endoligonucleotide” or “transferred strand” and the “non-transferred strandend oligonucleotide,” or “non-transferred strand” which, in combination,exhibit the sequences of the transposon end, and in which one or bothstrand comprise additional sequence.

The terms “transferred transposon end oligonucleotide” and “transferredstrand” are used interchangeably and refer to the transferred portion ofboth “transposon ends” and “transposon end compositions,” i.e.,regardless of whether the transposon end is attached to a tag or othermoiety. Similarly, the terms “non-transferred transposon endoligonucleotide” and “non-transferred strand” are used interchangeablyand refer to the non-transferred portion of both “transposon ends” and“transposon end compositions.” “In some embodiments, a transposon endcomposition is a “hairpin transposon end composition.” As used herein, a“hairpin transposon end composition.” means a transposon end compositionconsisting of a single oligodeoxyribonucleotide that exhibits anon-transferred transposon end sequence at its 5′-end, a transferredtransposon end sequence at its 3′-end, and an intervening arbitrarysequence between the non-transferred transposon end sequence and thetransferred transposon end sequence that is sufficiently long to allowintramolecular stem-loop formation, such that the transposon end portioncan function in a transposition reaction. In some embodiments, the5′-end of the hairpin transposon end composition has a phosphate groupin the 5′-position of the 5′-nucleotide. In some embodiments, theintervening arbitrary sequence between the non-transferred transposonend sequence and the transferred transposon end sequence of a hairpintransposon end composition provides a tag (e.g., including one or moretag domains) for a particular use or application.

In some embodiments, the methods of the present invention produce taggedcircular ssDNA fragments. In some embodiments, tagged circular ss DNAfragments exhibit only the sequence of the transferred strand of thetransposon end composition, and the tagged circular ssDNA fragments donot exhibit the sequence of the non-transferred strand of the transposonend composition.

In some embodiments, the transposon end composition used in the methodof the present invention comprises transposon end oligonucleotides thatexhibit only the transposon end sequences that form a complex with thetransposase or integrase and that are needed for the transpositionreaction; in these embodiments, the tag in the tagged circular ssDNAfragments generated using the method exhibits only the transferredtransposon end sequence.

However, in some embodiments, the transposon end composition comprisesor consists of at least one transposon end oligonucleotide that exhibitsone or more other nucleotide sequences in addition to the transposon endsequences. Thus, in some embodiments, the transposon end compositioncomprises a transferred strand that exhibits one or more othernucleotide sequences 5′-of the transferred transposon end sequence,which one or more other nucleotide sequences are also exhibited by thetag. Thus, in addition to the transferred transposon end sequence, thetag can have one or more other tag portions or tag domains.

As used herein, a “tag portion” or a “tag domain” means a portion ordomain of a tag that exhibits a sequence for a desired intended purposeor application. One tag portion or tag domain is the “transposon enddomain,” which tag portion or tag domain exhibits the transferredtransposon end sequence. In some embodiments wherein the transferredstrand also exhibits one or more other nucleotide sequences 5′-of thetransferred transposon end sequence, the tag also has one or more other“tag domains” in said 5′-portion, each of which tag domains is providedfor any desired purpose. For example, some embodiments of the inventioncomprise or consist of a transposon end composition that comprises orconsists of: (i) a transferred strand that exhibits one or moresequences 5′-of the transferred transposon end sequence that comprisesor consists of a tag domain selected from among one or more of arestriction site tag domain, a capture tag domain, a sequencing tagdomain, an amplification tag domain, a detection tag domain, an addresstag domain, and a transcription promoter domain; and (ii) anon-transferred strand that exhibits the non-transferred transposon endsequence. The invention comprises embodiments of the method that use anyone or more of said transposon end compositions.

As used herein an “cleavage domain” refers to a nucleic acid sequencethat is susceptible

As used herein, a “restriction site domain” means a tag domain thatexhibits a sequence for the purpose of facilitating cleavage using arestriction endonuclease. For example, in some embodiments, therestriction site domain is used to generate di-tagged linear ssDNAfragments. In some embodiments, the restriction site domain is used togenerate a compatible double-stranded 5′-end in the tag domain so thatthis end can be ligated to another DNA molecule using atemplate-dependent DNA ligase. In some preferred embodiments, therestriction site domain in the tag exhibits the sequence of arestriction site that is present only rarely, if at all, in the targetDNA (e.g., a restriction site for a rare-cutting restrictionendonuclease such as NotI or AscI). In some preferred embodiments, therestriction site in the restriction site domain is for a type IIrestriction endonuclease, such as FokI restriction endonuclease.

In some embodiments wherein the transferred strand of the transposon endcomposition comprises one or more restriction site domains 5′-of thetransferred transposon end sequence, the method further comprises:annealing an oligodeoxyribonucleotide that is complementary to thesingle-stranded restriction site of the tagged circular ssDNA fragmentsand then cleaving the tagged circular ssDNA fragments at the restrictionsite using the restriction endonuclease that recognizes the restrictionsite. Thus, in some embodiments, the method comprises linearizing thetagged circular ssDNA fragments to generate di-tagged linear ssDNAfragments.

In some other embodiments wherein the transferred strand of thetransposon end composition comprises one or more restriction sitedomains 5′-of the transferred transposon end sequence, the transferredstrand of the transposon end composition comprises a double-strandedhairpin comprising the restriction site, and the method furthercomprises the steps of cleaving the tagged linear ssDNA fragments at therestriction site using the restriction endonuclease that recognizes therestriction site; however, in some embodiments, this method is notpreferred because the double-stranded hairpin provides a site of dsDNAinto which the transposon end composition can be transposed by thetransposase or integrase.

In some preferred embodiments comprising (i) generating adouble-stranded restriction site, either by annealing of anoligodeoxyribonucleotide that is complementary to the single-strandedrestriction site, or by using a transferred strand that comprises adouble-stranded hairpin, and (ii) then cleaving the restriction siteusing the restriction endonuclease that recognizes the double-strandedrestriction site, the method further comprises the step of ligating therestriction endonuclease-cleaved tagged linear ssDNA fragments toanother DNA molecule that has a compatible 3′-end.

As used herein, a “capture tag domain” or a “capture tag” means a tagdomain that exhibits a sequence for the purpose of facilitating captureof the ssDNA fragment to which the tag domain is joined (e.g., toprovide an annealing site or an affinity tag for a capture of the taggedcircular ssDNA fragments or the di-tagged linear ssDNA fragments on abead or other surface, e.g., wherein the annealing site of the tagdomain sequence permits capture by annealing to a specific sequencewhich is on a surface, such as a probe on a bead or on a microchip ormicroarray or on a sequencing bead). In some embodiments of the method,after the tagged circular ssDNA fragments or the di-tagged linear ssDNAfragments are captured by annealing to a complementary probe on asurface, the capture tag domain provides a site for priming DNAsynthesis using said tagged circular ssDNA fragments or said di-taggedlinear ssDNA fragments (or the complements of said tagged circular ssDNAfragments or di-tagged linear ssDNA fragments) as templates. In someother embodiments, the capture tag domain comprises a 5′-portion of thetransferred strand that is joined to a chemical group or moiety thatcomprises or consists of an affinity binding molecule (e.g., wherein the5′-portion of the transferred strand is joined to a first affinitybinding molecule, such as biotin, streptavidin, an antigen, or anantibody that binds the antigen, that permits capture of the circulartagged ssDNA fragments or the di-tagged linear ssDNA fragments on asurface to which a second affinity binding molecule is attached thatforms a specific binding pair with the first affinity binding molecule).

As used herein, a “sequencing tag domain” or a “sequencing tag” means atag domain that exhibits a sequence for the purposes of facilitatingsequencing of the ssDNA fragment to which the tag is joined using themethod to synthesize tagged circular ssDNA fragments (e.g., to provide apriming site for sequencing by synthesis, or to provide annealing sitesfor sequencing by ligation, or to provide annealing sites for sequencingby hybridization). For example, in some embodiments, the sequencing tagdomain provides a site for priming DNA synthesis of said ssDNA fragmentor the complement of said ssDNA fragment.

As used herein, an “amplification tag domain” means a tag domain thatexhibits a sequence for the purpose of facilitating amplification of anucleic acid to which said tag is appended. For example, in someembodiments, the amplification tag domain provides a priming site for anucleic acid amplification reaction using a DNA polymerase (e.g., a PCRamplification reaction or a strand-displacement amplification reaction,or a rolling circle amplification reaction), or a ligation template forligation of probes using a template-dependent ligase in a nucleic acidamplification reaction (e.g., a ligation chain reaction).

As used herein, a “detection tag domain” or a “detection tag” means atag domain that exhibits a sequence or a detectable chemical orbiochemical moiety for the purpose of facilitating detection of thetagged circular ssDNA fragments or the di-tagged linear ssDNA fragments(e.g., wherein the sequence or chemical moiety comprises or is joined toa detectable molecule; such as a detectable molecule selected fromamong: a visible, fluorescent, chemiluminescent, or other detectabledye; an enzyme that is detectable in the presence of a substrate, e.g.,an alkaline phosphatase with NBT plus BCIP or a peroxidase with asuitable substrate); a detectable protein, e.g., a green fluorescentprotein; and an affinity-binding molecule that is bound to a detectablemoiety or that can form an affinity binding pair or a specific bindingpair with another detectable affinity-binding molecule; or any of themany other detectable molecules or systems known in the art).

As used herein, an “address tag domain” or an “address tag” means a tagdomain that exhibits a sequence that permits identification of aspecific sample (e.g., wherein the transferred strand has a differentaddress tag domain that exhibits a different sequence for each sample).

As used herein, a “transcription promoter domain” or a “promoter domain”means a tag domain that exhibits a sequence for a sense promotersequence or for an anti-sense promoter sequence of an RNA polymerasepromoter. As used herein, a “sense promoter sequence” means the sequenceof an RNA polymerase promoter that is joined to the DNA strand thatserves as the template for transcription by an RNA polymerase whichbinds the RNA polymerase promoter and initiates transcription therefromunder reaction conditions suitable for transcription. As used herein, an“anti-sense promoter sequence” means the sequence of an RNA polymerasepromoter that is complementary to the sense promoter sequence. In someembodiments, the sense promoter sequence exhibited by the transcriptionpromoter domain is for an RNA polymerase that binds a single-strandedRNA polymerase promoter and initiates transcription therefrom, in whichembodiments the sense promoter sequence is sufficient to function as theRNA polymerase promoter (e.g., for bacteriophage N4 RNA polymerase). Insome embodiments, the sense promoter sequence is for an RNA polymerasethat binds a double-stranded RNA polymerase promoter and initiatestranscription therefrom, in which embodiments the method comprisesmaking the RNA polymerase promoter double-stranded (e.g., by annealingto the sense promoter sequence an oligodeoxyribonucleotide that exhibitsan anti-sense promoter sequence that is complementary to the sensepromoter sequence, or by using the tagged circular ssDNA fragments orthe di-tagged linear ssDNA fragments as templates for synthesis of dsDNAcomprising or consisting of the sense promoter sequence) prior totranscription using an RNA polymerase that binds to and initiatestranscription from the double-stranded RNA polymerase promoter. In someembodiments, the sense promoter sequence is for a T7-type RNA polymerase(e.g., selected from among T7 RNA polymerase, T3 RNA polymerase, and SP6RNA polymerase). A transcription promoter domain that exhibits a sensepromoter sequence enables synthesis of RNA that is complementary to thesingle-stranded target DNA to which the transferred strand of thetransposon end composition is ligated using the method. Tagged circularssDNA fragments generated using a transposon end composition comprisinga transferred strand that has a transcription promoter domain thatexhibits an anti-sense promoter sequence cannot be transcribed by an RNApolymerase. However, in some embodiments, dsDNA synthesized by extendinga primer that anneals to the tagged circular ssDNA fragments is used fortranscription by an RNA polymerase that binds to and initiatestranscription from a double-stranded RNA polymerase promoter; in theseembodiments, the RNA synthesized exhibits the same sequence as thetagged circular ssDNA fragments.

The names and descriptions of different tag domains are for convenience,such as to make it easier to understand and discuss the intendedpurposes and applications of the different portions or domains of thetag in different embodiments. However, these names and descriptions arenot intended to limit the use or applications of the tag or of any ofits tag domains in any way. Thus, any particular tag or tag domain canbe used for any purpose in addition to, or in place of the intended orprimary purpose or application. Also, one tag domain can comprise two ormore other tag domains (e.g., a sequencing tag domain can comprise boththe transposon end domain and another tag domain 5′-of the transposonend domain) or one tag domain can provide the functions or purposes orapplications of two or more different tag domains (e.g., the transposonend domain can provide the purpose of the transferred transposon end andalso provide the function or purpose of a sequencing tag domain and/or acapture tag domain for a particular application). Still further, the tagneed not be described in terms of one or more different domains in orderto be used for any particular purpose or application or function.

As used herein, the terms “amplify” or “amplified” “amplifying” as usedin reference to a nucleic acid or nucleic acid reactions, refer to invitro methods of making copies of a particular nucleic acid, such as atarget nucleic acid, or a tagged nucleic acid produced, for example, byan embodiment of the present invention. Numerous methods of amplifyingnucleic acids are known in the art, and amplification reactions includepolymerase chain reactions, ligase chain reactions, strand displacementamplification reactions, rolling circle amplification reactions,transcription-mediated amplification methods such as NASBA (e.g., U.S.Pat. No. 5,409,818), loop mediated amplification methods (e.g., “LAMP”amplification using loop-forming sequences, e.g., as described in U.S.Pat. No. 6,410,278). The nucleic acid that is amplified can be DNAcomprising, consisting of, or derived from DNA or RNA or a mixture ofDNA and RNA, including modified DNA and/or RNA. The products resultingfrom amplification of a nucleic acid molecule or molecules (i.e.,“amplification products”), whether the starting nucleic acid is DNA, RNAor both, can be either DNA or RNA, or a mixture of both DNA and RNAnucleosides or nucleotides, or they can comprise modified DNA or RNAnucleosides or nucleotides. A “copy” does not necessarily mean perfectsequence complementarity or identity to the target sequence. Forexample, copies can include nucleotide analogs such as deoxyinosine ordeoxyuridine, intentional sequence alterations (such as sequencealterations introduced through a primer comprising a sequence that ishybridizable, but not complementary, to the target sequence, and/orsequence errors that occur during amplification.

“Affinity binding substances” or “affinity binding molecules” or“affinity molecules” herein means molecules that have affinity for and“bind” to each other under certain conditions, referred to as “bindingconditions”, to form a “specific binding pair.” For example, biotin andstreptavidin, biotin and avidin, or digoxigenin and a specific antibodythat binds digoxigenin are examples of “specific binding pairs,” withthe members of each specific binding pair comprising “affinity bindingmolecules” or “affinity binding substances” or “affinity molecules.”Affinity binding molecules (e.g., biotin and/or streptavidin) can becovalently joined or conjugated, or non-covalently bound, to othermolecules (e.g., to RNA or DNA) or to a solid surface using methodsknown in the art (e.g., using reagents and methods as described inAvidin-Biotin Chemistry: A Handbook, by D. Savage et al., PierceChemical Company, 1992, and in Handbook of Fluorescent Probes andResearch Products, Ninth Edition, by R. P. Hoagland, Molecular Probes,Inc., and in BIOCONJUGATE Techniques, by Greg T. Hermanson, Published byAcademic Press, Inc., San Diego, Calif., 1996). Affinity molecules thatare conjugated to DNA or RNA can also be synthesized using anoligonucleotide synthesizer using reagents and methods known in the art.

The term “binding” according to the present invention means theinteraction between an affinity molecule and an affinity bindingsubstance as a result of non-covalent bonds, such as, but not limitedto, hydrogen bonds, hydrophobic interactions, van der Waals bonds, andionic bonds. Without being bound by theory, it is believed in the artthat these kinds of non-covalent bonds result in binding, in part due tocomplementary shapes or structures of the molecules involved in thespecific binding pair. Based on the definition for “binding,” and thewide variety of affinity binding molecules or specific binding pairs, itis clear that binding conditions vary for different specific bindingpairs. Those skilled in the art can easily find or determine conditionswhereby, in a sample, binding occurs between the affinity bindingmolecules. In particular, those skilled in the art can easily determineconditions whereby binding between affinity binding molecules that wouldbe considered in the art to be “specific binding” can be made to occur.As understood in the art, such specificity is usually due to the higheraffinity between the affinity binding molecules than for othersubstances and components (e.g., vessel walls, solid supports) in asample. In certain cases, the specificity might also involve, or mightbe due to, a significantly more rapid association of affinity bindingmolecules than with other substances and components in a sample.

The terms “anneal” or “hybridize” and “annealing” or “hybridization”refer to the formation of complexes between nucleotide sequences thatare sufficiently complementary to form complexes via Watson-Crick basepairing. With respect to the present invention, nucleic acid sequencesthat are “complementary to” or “complementary with” or that “hybridize”or “anneal” to or with each other should be capable of forming or form“hybrids” or “complexes” that are sufficiently stable to serve theintended purpose. It is not required that every nucleic acid base withina sequence exhibited by one nucleic acid molecule is capable ofbasepairing or is paired with or is complexed with every nucleic acidbase within a sequence exhibited by a second nucleic acid molecule inorder for the two nucleic acid molecules or the respective sequencesexhibited therein to be “complementary” or “annealed” or “hybridized” toor with each other. As used herein, the terms “complementary” or“complementarity” are used in reference to a sequence of nucleotidesrelated by the base-pairing rules. For example, the sequence5′-A-G-T-3′, is complementary to the sequence 3′-T-C-A-S′.Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponhybridization of nucleic acids. The term “homology” refers to a degreeof complementarity of one nucleic acid sequence with another nucleicacid sequence. There may be partial homology or complete homology (i.e.,complementarity). A partially complementary sequence is one that atleast partially inhibits a completely complementary sequence fromhybridizing to a target nucleic acid and is referred to using thefunctional term “substantially homologous.” The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks complementarity orthat has only a low degree of complementarity (e.g., less than about 30%complementarity). In the case in which specific binding is low ornon-existent, the probe will not hybridize to a nucleic acid target.When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or a genomic clone, the term “substantially homologous” refersto any oligonucleotide or probe which can hybridize to either or bothstrands of the double-stranded nucleic acid sequence under conditions oflow stringency as described herein. As used herein, the terms“annealing” or “hybridization” are used in reference to the pairing ofcomplementary nucleic acid strands. Hybridization and the strength ofhybridization (i.e., the strength of the association between nucleicacid strands) is impacted by many factors well known in the artincluding the degree of complementarity between the nucleic acids,stringency of the conditions involved affected by such conditions as theconcentration of salts, the T_(m) (melting temperature) of the formedhybrid, the presence of other components (e.g., the presence or absenceof polyethylene glycol or betaine), the molarity of the hybridizingstrands and the G:C content of the nucleic acid strands.

In general, “cDNA” or a “cDNA molecule” refers to “complementary DNA”that is synthesized by RNA-dependent DNA polymerase- or reversetranscriptase-catalyzed extension of a primer that anneals to an RNAmolecule of interest using at least a portion of the RNA molecule ofinterest as a template (which process is also called “reversetranscription”). The cDNA molecules synthesized are “homologous to” or“base pair with” or “form a complex with” at least a portion of thetemplate.

A as used herein, a “population of DNA fragments” refers to a pluralityor collection of DNA fragments, e.g., from a target DNA. In someembodiments a population of DNA fragments comprises a DNA fragmentlibrary comprising sequences that are qualitatively and/orquantitatively representative of the sequence of the target DNA, whilein some embodiments, a population of DNA fragments contains a subset ofa DNA library, e.g., it may not be representative of the sequence of thetarget DNA.

As used herein, a “DNA fragment library” or a “library of DNA fragments”means a collection or population of tagged DNA fragments (e.g.,di-tagged DNA fragments or tagged circular ssDNA fragments) generatedfrom target DNA, wherein the combination of the tagged DNA fragments inthe collection or population exhibits sequences that are qualitativelyand/or quantitatively representative of the sequence of the target DNAfrom which the tagged DNA fragments were generated, and wherein thetagged DNA fragments that are in the collection or population have notbeen selected for or selected against by intentionally using a methodthat either includes or excludes tagged DNA fragments based on thenucleotide or sequence composition of the target DNA. For a variety ofreasons, it is possible that a DNA fragment library may not contain atagged DNA fragment representing every sequence which is exhibited bythe target DNA. For example, in some embodiments, the tagged DNAfragment library may not contain tagged DNA fragments that exhibitsequences of the ends of a target DNA comprising linear dsDNA (e.g., dueto a low frequency of insertion of two transposon end compositions intothe end portions of the target DNA). Generally, a lower frequency orlack of tagged DNA fragments that exhibit sequences of certain portionsor regions of the target DNA is acceptable for the intended purpose orapplication. However, the invention also comprises additional methodembodiments for those situations when it is considered important ordesirable for a particular purpose or application to generate a DNAfragment library wherein there is a higher probability that the taggedDNA fragments exhibit every sequence which is exhibited by the targetDNA from which the fragments were generated (e.g., see the section ofthe specification entitled (“Methods for Generating DNA FragmentLibraries with Improved Representation of Sequences at the Ends of theTarget DNA”). Still further, in some cases the probability that the DNAfragment library will contain a tagged DNA fragment that exhibits everysequence of the target DNA will be increased if more molecules of targetDNA are present in the transposition reaction step of the method,thereby generating more molecules of 5′-tagged DNA fragments using themethod. Thus, still another method for increasing the probability that aDNA fragment library will contain a tagged DNA fragment that exhibitsevery sequence which is exhibited by the target DNA is to amplify thetarget DNA and then use the amplified target DNA in place of the targetDNA for generating the DNA fragment library. In still other embodimentswherein target DNA comprises dsDNA prepared from RNA using a reversetranscription reaction, the amount of target DNA is amplified byamplifying the RNA prior to converting it to dsDNA using the reversetranscription step. Some methods for amplification of RNA and DNAmolecules that can be used for providing amplified target DNA aredisclosed herein. However, the invention is not limited with respect tothe method used for amplifying the target DNA. In some embodiments, thetarget DNA is amplified using one of the methods disclosed herein,whereas in some other embodiments, another method known in the art isused.

As used herein, the term “nucleic acid modifying enzyme” refers to anyenzyme that acts upon DNA to effect a modification, e.g., cleavage,ligation, polymerization, phosphorylation, etc. Nucleic acid modifyingenzymes include, e.g., polymerases, nucleases, transferrases, ligases,phosphorylases, phosphatases, methylases, transosases, etc. “DNAmodifying enzymes” comprise any enzymes that act on DNA includingenzymes that also act on other substrates, such as RNA.

As used herein, a “DNA polymerase” refers to an enzyme that catalyzesthe polymerization of deoxyribonucleotides into a DNA strand. DNApolymerases comprise “template-dependent DNA polymerases,” which requirea template nucleic acid to determine the order in whichdeoxyribonucleotides are added in the polymer, or they may be“template-independent” such that they catalyze polymerization withoutreference to a template sequence.

A “DNA-dependent DNA polymerase” is an enzyme that synthesizes acomplementary DNA (“cDNA”) copy by extension of a primer that isannealed to a DNA template. Some DNA-dependent DNA polymerases may alsosynthesize a complementary DNA copy from an RNA template, a process thatis also referred to as “reverse transcription.” DNA polymerases that canreverse-transcribe can also be referred to as a “reversetranscriptases.”

In addition to synthesizing DNA polymers, DNA polymerases may compriseother features or activities. For example, a DNA polymerase may becharacterizes as having or lacking 5′ to 3′ exonuclease activity (alsoreferred to a 5′ exonuclease or 5′ nuclease activity), 3′ to 5′exonuclease activity, strand displacement activity, and they may becharacterized with respect to the degree they are processive ordistributive, as discussed in more detail below.

Some DNA polymerases are able to displace the strand complementary tothe template strand as a new DNA strand is synthesized by thepolymerase. This process is called “strand displacement” and the DNApolymerases that have this activity are referred to herein as“strand-displacing DNA polymerases.” The template for stranddisplacement DNA synthesis can be a linear or circular single-strandedDNA (ssDNA) or double-stranded DNA (dsDNA). If the DNA template is asingle-stranded circle, primed DNA synthesis proceeds around and aroundthe circle, with continual displacement of the strand ahead of thereplicating strand, a process called “rolling circle replication.”Rolling circle replication results in synthesis of tandem copies of thecircular template. In general, it is preferred that aDNA-template-specific DNA polymerase used for a method of the inventionefficiently synthesizes DNA of a suitable length for the intendedpurpose without “falling off” of the template (or terminating synthesisof the DNA), which is referred to as the enzyme's processivity. Thecapability of a DNA polymerase to strand displace can be readilydetermined using the polymerase in a rolling circle replication assay asdescribed by Fire and Xu (Proc. Natl. Acad. Sci. USA 92: 4641-4645,1995). Strand displacement and DNA polymerase processivity can also beassayed using methods described in Kong et al. (J. Biol. Chem. 268:1965-1975, 1993). Terminal transferase is also defined as a DNApolymerase herein, which DNA polymerase is used as a composition in someembodiments of the kits and methods of the present invention. Terminaltransferase is preferred in some embodiments because it catalyzestemplate-independent addition of dNTPs to the 3′-hydroxyl termini ofDNA.

Some embodiments comprise a method that uses a DNA polymerasecomposition that has 5′-to-3′ exonuclease activity to release anucleotide that is labeled with a detectable moiety (e.g., a moietycomprising a visible, fluorescent, chemiluminescent, or other detectablemolecule) as a means for assaying DNA polymerization, and thereby,detecting and/or quantifying the presence in the sample of the nucleicacid molecule that serves as the template (e.g., in a manner similar tothe TaqMan® assays of Applied Biosystems, Inc.). In some embodiments,the present invention comprises a DNA polymerase composition that lacks5′-to-3′ exonuclease activity.

Some embodiments comprise a method that uses a DNA polymerasecomposition that lacks 5′-to-3′ exonuclease activity. For example, insome embodiments, a DNA polymerase composition that lacks 5′-to-3′exonuclease activity is used for DNA sequencing. For example, in someother embodiments, a DNA polymerase composition that lacks 5′-to-3′exonuclease activity is used for whole genome amplification.

In some embodiments, the present invention comprises a DNA polymerasecomposition that has 5′-to-3′ exonuclease activity. In some preferredembodiments (e.g., wherein a DNA polymerase is used, in addition to atemplate-dependent ligase, for joining in one of the methods describedherein), the method uses a DNA polymerase composition that lacks 5′nuclease activity (including both 5′-to-3′ exonuclease and 5′structure-dependent nuclease activity). For example, in some otherembodiments, a DNA polymerase composition that lacks 5′-to-3′exonuclease activity is used for to fill a gap. Thus, in someembodiments of methods or kits, the present invention comprises a DNApolymerase composition that lacks 5′ nuclease activity. However, a DNApolymerase composition that has 5′ nuclease activity to release anucleotide or an oligonucleotide that is labeled with a detectablemoiety (e.g., a moiety comprising a visible, fluorescent,chemiluminescent, or other detectable molecule) as a means for assayingDNA polymerization, and thereby, detecting and/or quantifying thepresence in the sample of the nucleic acid molecule that serves as thetemplate (e.g., in a manner similar to the TaqMan® assays of AppliedBiosystems, Inc.) could be used for quantifying DNA molecules generatedusing a method of the invention.

Examples of strand-displacing DNA polymerases that can be used include,but are not limited to, RepliPHI™ phi29 DNA polymerase, DisplaceAce™ DNApolymerase, rGka DNA polymerase, SequiTherm™ DNA polymerase, Taq DNApolymerase, Tfl DNA polymerase, and MMLV reverse transcriptase (allavailable from EPICENTRE Biotechnologies, Madison, Wis., USA). In someembodiments, a blend of a DNA polymerase that lacks 3′-to-5′ exonucleaseproofreading activity with a DNA polymerase that has this activity, suchas FAILSAFE™ DNA polymerase is used as the strand-displacing DNApolymerase. The enzyme blend is useful in some embodiments because itexhibits improved fidelity during DNA synthesis (i.e., it synthesizesDNA with fewer nucleotides that are not complementary to the template).Fidelity and/or error rates of many DNA polymerases under particularconditions are known, as are methods for measuring fidelity (e.g., bysequencing).

In general, it is desirable in a strand-displacement amplificationmethod of the present invention that the amount of strand-displacing DNApolymerase used in the method is as high as possible without inhibitingor adversely affecting the reaction. For example, REPLIPHI™ phi29 DNApolymerase (EPICENTRE) can be used at about one microgram of protein ina 20-microliter reaction and DISPLACE™ DNA polymerase (EPICENTRE) can beused at about 50 units to about 300 units in a 50-microliter reaction.Since definitions for units vary for different DNA polymerases and evenfor similar DNA polymerases from different vendors or sources, and alsobecause the activity for each enzyme varies at different temperaturesand under different reaction conditions, it is desirable to optimize theamount of strand-displacing DNA polymerase and reaction conditions foreach DNA template and primer used.

Strand displacement can be facilitated through the use of a stranddisplacement factor, such as helicase, but since a variety of DNApolymerases can be used for the present invention, such a stranddisplacement factor is not usually required. It is considered that anyDNA polymerase that can perform rolling circle replication in thepresence of a strand displacement factor is suitable for use inembodiments of the invention that comprise strand displacement even ifthe DNA polymerase does not perform rolling circle replication in theabsence of such a factor. Strand displacement factors that permitrolling circle replication include, but are not limited to, BMRF1polymerase accessory subunit (Tsurumi et al., J. Virology, 67:7648-7653, 1993), adenovirus DNA-binding protein (Zijderveld and van derVliet, J. Virology, 68: 1158-1164, 1994), herpes simplex viral proteinICP8 (Boehmer and Lehman, J. Virology, 67: 711-715, 1993); Skaliter andLehman, Proc. Natl. Acad. Sci. USA, 91: 10,665-10,669, 1994),single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol.Chem., 270: 8910-8919, 1995), and calf thymus helicase (Siegel et al.,J. Biol. Chem., 267: 13,629-13,635, 1992), all of which are incorporatedherein by reference.

A “mononucleoside” or “nucleoside”, as used herein, refers to a compoundconsisting of a purine (guanine (G) or adenine (A)) or pyrimidine(thymine (T), uridine (U), or cytidine (C)) base covalently linked to apentose sugar, whereas “nucleotide” refers to a nucleosidephosphorylated at one of the hydroxyl groups of the pentose sugar. Theterm “canonical” is used to refer to the four common nucleic acid basesadenine, cytosine, guanine and thymine that are commonly found in DNA orto the respective deoxyribonucleosides, deoxyribonucleotides or2′-deoxyribonucleoside-5′-triphosphates that contain a canonical base.The term “non-canonical” is used to refer to nucleic acid bases in DNAother than the four canonical bases, or to the respectivedeoxyribonucleosides, deoxyribonucleotides, or2′-deoxyribonucleoside-5′-triphosphates that contain a non-canonicalbase. For example, although uracil is a common nucleic acid base in RNA,uracil is a non-canonical base in DNA. “Non-canonical bases” are foundin nucleic acids as a result of incorporation of non-canonicalnucleotides (e.g., by synthesis using an oligonucleotide synthesizer orby synthesis using a DNA polymerase) or as a result of modification ofexisting bases (canonical or non-canonical).

A “nucleic acid” or “polynucleotide” means a polymer molecule comprisinga series of “mononucleosides,” also referred to as “nucleosides,” inwhich the 3′-position of the pentose sugar of one nucleoside is linkedby an internucleoside linkage, such as, but not limited to, aphosphodiester bond, to the 5′-position of the pentose sugar of the nextnucleoside. A nucleoside linked to a phosphate group is referred to as a“nucleotide.” The nucleotide that is linked to the 5′-position of thenext nucleotide in the series is referred to as “5′ of” or the “5′nucleotide” and the nucleotide that is linked to the 3′-position of the5′ nucleotide is referred to as “3′ of” or the “3′ nucleotide.” As usedherein, the terms “5′-of” and “3′-of” refer to the position ororientation of a particular chemical group, nucleotide, sequence ofnucleotides, or genetic element (e.g., an RNA polymerase promotersequence) relative to another chemical group, nucleotide, sequence ofnucleotides, or genetic element within a single strand of a nucleicacid. If a first nucleic acid sequence is 3′-of a second sequence on onestrand, the complement of the first sequence will be 5′-of thecomplement of the second sequence on the complementary strand. Thedescription of the invention will be understood with respect to therelative 5′ or 3′ position and orientation of a sequence or geneticelement within a particular nucleic acid strand.

Linear nucleic acid molecules are said to have a “5′-terminus” (5′ end)and a “3′-terminus” (3′ end) because nucleic acid phosphodiesterlinkages occur at the 5′ carbon and 3′ carbon of the sugar moieties ofthe substituent mononucleotides. The end of a polynucleotide at which anew linkage would be to a 5′ carbon is its 5′ terminal nucleotide. Theend of a polynucleotide at which a new linkage would be to a 3′ carbonis its 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

The pentose sugar of the nucleic acid can be ribose, in which case, thenucleic acid or polynucleotide is referred to as “RNA,” or it can be2′-deoxyribose, in which case, the nucleic acid or polynucleotide isreferred to as “DNA.” Alternatively, especially if the nucleic acid issynthesized chemically, the nucleic acid can be composed of both DNA andRNA mononucleotides. In both RNA and DNA, each pentose sugar iscovalently linked to one of four common or “canonical” nucleic acidbases (each also referred to as a “base”). Three of the predominantnaturally-occurring bases that are linked to the sugars (adenine,cytidine and guanine) are common for both DNA and RNA, while one base isdifferent; DNA has the additional base thymine, while RNA has theadditional base uridine. In some cases, uridine can be present as a basein DNA. Those in the art commonly think of a small polynucleotide as an“oligonucleotide.” The term “oligonucleotide” as used herein is definedas a molecule comprising of two or more deoxyribonucleotides orribonucleotides, preferably about 6 to 100 nucleotides, but there is nodefined limit to the length of an oligonucleotide. The exact size willdepend on many factors, which in turn depends on the ultimate functionor use of the oligonucleotide.

Also, for a variety of reasons, a nucleic acid or polynucleotide of theinvention may comprise one or more modified nucleic acid bases, sugarmoieties, or internucleoside linkages. By way of example, some reasonsfor using nucleic acids or polynucleotides that contain modified bases,sugar moieties, or internucleoside linkages include, but are not limitedto: (1) modification of the T_(m); (2) changing the susceptibility ofthe polynucleotide to one or more nucleases; (3) providing a moiety forattachment of a label; (4) providing a label or a quencher for a label;or (5) providing a moiety, such as biotin, for attaching to anothermolecule which is in solution or bound to a surface. For example, insome embodiments, an oligonucleotide, such as a primer, may besynthesized so that a random portion contains one or moreconformationally restricted ribonucleic acid analogs, such as, but notlimited to one or more ribonucleic acid analogs in which the ribose ringis “locked” with a methylene bridge connecting the 2′-O atom with the4′-C atom (e.g., as available from Exiqon, Inc. under the trademark of“LNA™”); these modified nucleotides result in an increase in the T_(m)or melting temperature by about 2 degrees to about 8 degrees centigradeper nucleotide monomer. If the T_(m) is increased, it might be possibleto reduce the number of random nucleotides in the random 3′-portion ofthe terminal tagging oligoribonucleotide. However, a modifiednucleotide, such as an LNA must be validated to function in the methodfor its intended purpose, as well as satisfying other criteria of themethod. For example, in some embodiments wherein an oligonucleotideprimer comprising ribonucleotides is used, one criterion for using themodified nucleotide in the method can be that the oligonucleotide thatcontains it can be digested by a single-strand-specific RNase.

In order to accomplish the goals of the invention, by way of example,the nucleic acid bases in the mononucleotides of one or more positionsof a polynucleotide or oligonucleotide may comprise guanine, adenine,uracil, thymine, or cytidine, or alternatively, one or more of thenucleic acid bases may comprise a modified base, such as, but notlimited to xanthine, allyamino-uracil, allyamino-thymidine,hypoxanthine, 2-aminoadenine, 5-propynyl uracil, 5-propynyl cytosine,4-thiouracil, 6-thioguanine, aza and deaza uracils, thymidines,cytosines, adenines, or guanines Still further, they may comprise anucleic acid base that is derivatized with a biotin moiety, adigoxigenin moiety, a fluorescent or chemiluminescent moiety, aquenching moiety or some other moiety. The invention is not limited tothe nucleic acid bases listed; this list is given to show an example ofthe broad range of bases which may be used for a particular purpose in amethod.

With respect to nucleic acids or polynucleotides of the invention, oneor more of the sugar moieties can comprise 2′-deoxyribose, oralternatively, one or more of the sugar moieties can be some other sugarmoiety, such as, but not limited to, ribose, or 2′-fluoro-2′-deoxyriboseor 2′-O-methyl-ribose, which provide resistance to some nucleases, or2′-amino-2′-deoxyribose or 2′-azido-2′-deoxyribose, which can be labeledby reacting them with visible, fluorescent, infrared fluorescent orother detectable dyes or chemicals having an electrophilic,photoreactive, alkynyl, or other reactive chemical moiety.

The internucleoside linkages of nucleic acids or polynucleotides of theinvention can be phosphodiester linkages, or alternatively, one or moreof the internucleoside linkages can comprise modified linkages, such as,but not limited to, phosphorothioate, phosphorodithioate,phosphoroselenate, or phosphorodiselenate linkages, which are resistantto some nucleases.

When referring to an oligonucleotide or a portion of an oligonucleotidethat exhibits a “random sequence”, we mean that the oligonucleotide orportion thereof is synthesized (e.g., using an oligonucleotidesynthesizer) using equal amounts of all four of the canonical nucleotidebases (A, G, C, and T or U) for very nucleotide position within therandom sequence portion. This method results in synthesis of a mixtureof oligonucleotides comprising (4 to the n power)+1 of differentoligonucleotides, where “n” equals the number of nucleotide positionswithin the random sequence portion. Thus, in these embodiments, theoligonucleotide comprises a mixture of many different oligonucleotides,representing all possible sequences for the random sequence portion.When referring to an oligonucleotide or a portion of an oligonucleotidethat exhibits a “semi-random sequence”, we mean that the semi-randomoligonucleotide or portion is synthesized (e.g., using anoligonucleotide synthesizer) wherein some nucleotide positions aresynthesized using equal amounts of all four of the canonical nucleotidebases (A, G, C, and T or U) (i.e., those positions are “random” asdescribed above) but one or more other positions within the semi-randomportion are synthesized using only one, two, or three, rather than allfour, of the canonical base nucleotides (i.e., A, C, G, and T or U). Insome embodiments, an oligonucleotide contains one or more nucleotideswith a “degenerate base”, by which we mean a nucleic acid base that iscapable of base-pairing with one or more nucleic acid bases other thanaccording to the standard base-pairing rules that A pairs with T or Uand G pairs with C, and a “degenerate nucleotide” is a nucleotide thatcontains a degenerate base. A “portion” or “region,” usedinterchangeably herein, of a polynucleotide or oligonucleotide(including a primer) is a contiguous sequence of 2 or more bases. Inother embodiments, a region or portion is at least about any of 1, 2, 3,5, 10, 15, 20, 25, 50, 75, or even more contiguous nucleotides.

A “primer” is an oligonucleotide (“oligo”), generally with a free 3′-OHgroup, that can be extended by a nucleic acid polymerase. For atemplate-dependent polymerase, generally at least the 3′-portion of theprimer oligo is complementary to a portion of a template nucleic acid,to which the oligo “binds” (or “complexes,” “anneals,” or “hybridizes”),by hydrogen bonding and other molecular forces, to the template to givea primer/template complex for initiation of synthesis by a DNApolymerase, and which is extended (i.e., “primer extended”) by theaddition of covalently bonded bases linked at its 3′-end which arecomplementary to the template in the process of DNA synthesis. Theresult is a primer extension product. Template-dependent DNA polymerases(including reverse transcriptases) generally require complexing of anoligonucleotide primer to a single-stranded template to initiate DNAsynthesis (“priming”), but RNA polymerases generally do not require aprimer for synthesis of RNA that is complementary to a DNA template(transcription).

A “single-strand-specific DNase” means a DNase that specifically digestssingle-stranded DNA, but that does not digest single-stranded RNA or RNAor DNA that is annealed to or complexed with complementary RNA or DNA,whether said complementary RNA or DNA is part of another nucleic acidmolecule (e.g., by intermolecular base-pairing) or a portion of the samenucleic acid molecule (e.g., by intramolecular base-pairing). Thesingle-strand-specific DNase can be an endonuclease or an exonuclease,so long as it is active in specifically digesting single-stranded DNA tomonomers or short oligodeoxyribonucleotides. In some preferredembodiments, oligodeoxyribonucleotides, including primers, are removedfrom the reaction mixture after step of the method in which they areused by digestion with a single-strand-specific DNase. Exonuclease I,exonuclease VII, and Rec J exonuclease are exemplarysingle-strand-specific DNases.

A “T7-type RNA polymerase” (RNAP) herein means T7 RNA polymerase (e.g.,see Studier, F W et al., pp. 60-89 in Methods in Enzymology, Vol. 185,ed. by Goeddel, DV, Academic Press, 1990) or an RNAP derived from a“T7-type” bacteriophage, meaning a bacteriophage that has a similargenetic organization to that of bacteriophage T7. The geneticorganization of all T7-type phages that have been examined has beenfound to be essentially the same as that of T7. Examples of T7-typebacteriophages according to the invention include, but are not limitedto Escherichia coli phages T3, phi I, phi II, W31, H, Y, A1, 122, cro,C21, C22, and C23; Pseudomonas putida phage gh-1; Salmonella typhimuriumphage SP6; Serratia marcescens phages IV; Citrobacter phage ViIII; andKlebsiella phage No. 11 (Hausmann, Current Topics in Microbiology andImmunology 75:77-109, 1976; Korsten et al., J. Gen. Virol. 43:57-73,1975; Dunn, et al., Nature New Biology 230:94-96, 1971; Towle, et al.,J. Biol. Chem. 250:1723-1733, 1975; Butler and Chamberlin, J. Biol.Chem. 257:5772-5778, 1982), as well as mutant forms of such RNAPs (e.g.,Sousa et al., U.S. Pat. No. 5,849,546; Padilla, R and Sousa, R, NucleicAcids Res., 15: e138, 2002; Sousa, R and Mukherjee, S, Prog Nucleic AcidRes Mol. Biol., 73: 1-41, 2003; Guillerez, J, et al., U.S. PatentApplication No. 20040091854). In preferred embodiments of the invention,the promoter used is a wild-type or mutant promoter sequence that isrecognized by a T7-type RNA polymerase. In some embodiments, thepromoter can be single-stranded, such as a pseudopromoter (e.g., Ohmichiet al., Proc. Natl. Acad. Sci. USA 99:54-59, 2002), or an N4 vRNAPpromoter, in which case the truncated protein comprising thetranscriptionally active 1,106-amino acid domain (corresponding to aminoacids 998-2103) of the N4 vRNAP (designated “mini-vRNAP”; EPICENTREBiotechnologies, Madison, Wis., USA) is used (Kazmierczak, K. M., etal., EMBO J., 21: 5815-5823, 2002).

As used herein, “target DNA” refers to any dsDNA of interest that issubjected to transposition, e.g., for generating a library of tagged DNAfragments (e.g., 5′- and 3′-tagged or di-tagged linear ssDNA or dsDNAfragments or tagged circular ssDNA fragments).

“Target DNA” can be derived from any in vivo or in vitro source,including from one or multiple cells, tissues, organs, or organisms,whether living or dead, or from any biological or environmental source(e.g., water, air, soil). For example, in some embodiments, the targetDNA comprises or consists of eukaryotic and/or prokaryotic dsDNA thatoriginates or that is derived from humans, animals, plants, fungi,(e.g., molds or yeasts), bacteria, viruses, viroids, mycoplasma, orother microorganisms. In some embodiments, the target DNA comprises orconsists of genomic DNA, subgenomic DNA, chromosomal DNA (e.g., from anisolated chromosome or a portion of a chromosome, e.g., from one or moregenes or loci from a chromosome), mitochondrial DNA, chloroplast DNA,plasmid or other episomal-derived DNA (or recombinant DNA containedtherein), or double-stranded cDNA made by reverse transcription of RNAusing an RNA-dependent DNA polymerase or reverse transcriptase togenerate first-strand cDNA and then extending a primer annealed to thefirst-strand cDNA to generate dsDNA. In some embodiments, the target DNAcomprises multiple dsDNA molecules in or prepared from nucleic acidmolecules (e.g., multiple dsDNA molecules in or prepared from genomicDNA or cDNA prepared from RNA in or from a biological (e.g., cell,tissue, organ, organism) or environmental (e.g., water, air, soil,saliva, sputum, urine, feces) source. In some embodiments, the targetDNA is from an in vitro source. For example, in some embodiments, thetarget DNA comprises or consists of dsDNA that is prepared in vitro fromsingle-stranded DNA (ssDNA) or from single-stranded or double-strandedRNA (e.g., using methods that are well-known in the art, such as primerextension using a suitable DNA-dependent and/or RNA-dependent DNApolymerase (reverse transcriptase). In some embodiments, the target DNAcomprises or consists of dsDNA that is prepared from all or a portion ofone or more double-stranded or single-stranded DNA or RNA moleculesusing any methods known in the art, including methods for: DNA or RNAamplification (e.g., PCR or reverse-transcriptase-PCR (RT-PCR),transcription-mediated amplification methods, with amplification of allor a portion of one or more nucleic acid molecules); molecular cloningof all or a portion of one or more nucleic acid molecules in a plasmid,fosmid, BAC or other vector that subsequently is replicated in asuitable host cell; or capture of one or more nucleic acid molecules byhybridization, such as by hybridization to DNA probes on an array ormicroarray (e.g., by “sequence capture”; e.g., using kits and/or arraysfrom ROCHE NIMBLEGEN, AGILENT, or FEBIT).

In some embodiments, “target DNA” means dsDNA that is prepared ormodified (e.g., using various biochemical or molecular biologicaltechniques) prior to being used for generating a library of tagged DNAfragments (e.g., 5′- and 3′-tagged or di-tagged linear ssDNA or dsDNAfragments or tagged circular ssDNA fragments). For example, the presentinventors observed that the representation of next-generation sequencedata from the ends of target DNA comprising dsDNA molecules with a sizeof less than 10 Kb was low compared to the representation of sequencedata from the middle of that target DNA. Without being bound by theory,one possible explanation for this observation is that the probability offinding DNA fragments with two transposon end compositions inserted inopposite orientations at the ends of a linear dsDNA molecule is lowerthan the probability of finding DNA fragments with two transposon endcompositions inserted in opposite orientations in the middle of thelinear dsDNA molecule. Thus, in some embodiments, in order to generatelibraries of di-tagged DNA fragments or tagged circular DNA fragmentsthat better represent the end sequences, the method further comprisesproviding target DNA for use in the method comprising dsDNA (e.g.,double-stranded genomic DNA or cDNA prepared from RNA, such as mRNA)that already has a tag on the 5′ and/or 3′ end. For example, in someembodiments, the target DNA comprises double-stranded cDNA that isprepared from RNA by: synthesizing first-strand cDNA by extending afirst-strand cDNA synthesis primer that has a 3′-portion and a5′-portion, wherein the 3′-portion is complementary to the 3′-endportion of the RNA and the 5′-portion comprises a first tag, thenjoining a second tag to the 3′-end of the first-strand cDNA using aterminal tagging oligonucleotide and a DNA polymerase as describedelsewhere herein, and then using a DNA polymerase to synthesizedouble-stranded cDNA by extending a second-strand cDNA synthesis primerthat anneals to the second tag. Alternatively, in some other preferredembodiments, in order to generate libraries of di-tagged DNA fragmentsthat better represent the end sequences, the target DNA used in themethod for generating di-tagged DNA fragments or tagged circular DNAfragments comprises circular dsDNA that is prepared by intramolecularligation of linear dsDNA (e.g., that is prepared by intramolecularligation of double-stranded genomic DNA or of double-stranded cDNAprepared from RNA, such as mRNA). Thus, in some embodiments, the methodfurther comprises: ligating the linear dsDNA using a ligase (e.g., T4DNA ligase) to generate circular dsDNA for use as target DNA in themethod. In some embodiments of the method comprising generating circulardsDNA for use as target DNA by ligating linear dsDNA, the linear dsDNAis treated with T4 DNA polymerase and T4 polynucleotide kinase (e.g.,using the END-It™ DNA End Repair Kit (EPICENTRE Biotechnologies,Madison, Wis., USA) prior to the ligation step in order to make the endsblunt and phosphorylate the 5′-ends.

As used herein, a “DNA fragment” means a portion or piece or segment ofa target DNA that is cleaved from or released or broken from a longerDNA molecule such that it is no longer attached to the parent molecule.A DNA fragment can be double-stranded (a “dsDNA fragment”) orsingle-stranded (a “ssDNA fragment”), and the process of generating DNAfragments from the target DNA is referred to as “fragmenting” the targetDNA. In some preferred embodiments, the method is used to generate a“DNA fragment library” comprising a collection or population of taggedDNA fragments.

A “template” is a nucleic acid molecule that is being copied by anucleic acid polymerase, such as a DNA polymerase. Whether the nucleicacid molecule comprises two strands (i.e., is “double-stranded”) or onlyone strand (i.e., is “single-stranded”), the strand of said nucleic acidmolecule that serves to specify the sequence of nucleotides exhibited bya nucleic acid that is synthesized is the “template” or “the templatestrand.” The nucleic acid synthesized by the nucleic acid polymerase iscomplementary to the template. Both RNA and DNA are always synthesizedin the 5′-to-3′ direction, beginning at the 3′-end of the templatestrand, and the two strands of a nucleic acid duplex always are alignedso that the 5′ ends of the two strands are at opposite ends of theduplex (and, by necessity, so then are the 3′ ends). A primer isrequired for both RNA and DNA templates to initiate synthesis by a DNApolymerase, but a primer is not required to initiate synthesis by aDNA-dependent RNA polymerase, which is usually called simply an “RNApolymerase.”

“Terminal transferase”, also referred to as “terminaldeoxyribonucleotidyl transferase” or “TdT”, is a DNA polymerase thatcatalyzes template-independent addition (or “tailing”) ofdeoxyribonucleoside triphosphates (dNTPs) or a singledideoxyribonucleoside triphosphate to the 3′-hydroxyl termini of DNA. Acommon terminal transferase used in the art, which is commerciallyavailable, is produced in an E. coli strain that expresses therecombinant gene from calf thymus. In some embodiments, the inventionfurther comprises the step of incubating 5′-tagged DNA fragments, afterdenaturation, with TdT and a dNTP under conditions and for sufficienttime wherein the 5′- and 3′-tagged DNA fragments that have a second tagcomprising a homopolymeric DNA tail is synthesized. In some embodiments,the homopolymeric DNA tail is further used as a priming site forsynthesis of double-stranded cDNA. In some embodiments, the primer usedfor synthesizing the second strand of DNA has a 3′-portion that iscomplementary to the second tag comprising the homopolymeric tail and a5′-portion that exhibits an arbitrary desired sequence that is notcomplementary to the first tag, the target DNA or the second tagcomprising the homopolymeric tail. For example, in some embodiments, the5′-portion of the primer exhibits an anti-sense promoter sequence for anRNA polymerase promoter and the method further comprise incubating theresulting double-stranded cDNA with the RNA polymerase under conditionsand for sufficient time wherein RNA is synthesized.

In some embodiments, the transposon end oligonucleotides used in themethod of the present invention exhibit only the transposon endsequences needed in a transposition reaction. However, in someembodiments, at least one of the transposon end oligonucleotidesadditionally exhibits one or more other nucleotide sequences 5′-of thetransposon end sequence. Thus, in some embodiments, the method or kituses a transferred strand that has a 3′ portion and a 5′ portion,wherein the 3′ portion exhibits the transferred transposon end sequenceand the 5′ portion exhibits one or more additional sequences that do notparticipate in forming a functional complex with the transposase. Thereis no limit to which additional sequences are used for the one or moreadditional sequences in the 5′-portion of the transferred strand, whichsequences can be used to accomplish any desired purpose. For example, insome embodiments, the 5′ portion of the transferred strand exhibits oneor more additional tag sequences (e.g., a tag sequence that permitscapture by annealing to a specific sequence on a surface, such as a beador a probe on a microchip or array; e.g., for capture on a bead fornext-generation sequencing; e.g., a 454A or 454B tag sequence forcapture on the bead for sequencing using a Roche 454 Next-Gen sequencer)or one or more sequences for identification, detection (e.g.,fluorescent detection), or sorting of the products of the method. Insome other embodiments, the 5′ portion of the transferred strandexhibits one or more additional nucleotides or sequences or a chemicalgroup or moiety that comprises or consists of an affinity-binding that(e.g., a tag sequence that permits capture by annealing to a specificsequence on a surface, such as a bead or a probe on a microchip orarray. In some preferred embodiments, the size of the one or moreadditional sequences in the 5′-portion of the transferred strand areminimized in order to minimize the probability or frequency of insertionof the transferred strand into itself during the in vitro transposasereaction. For example, in some embodiments, the size of the 5′-portionof the transferred strand is less than about 150 nucleotides, less thanabout 100 nucleotides, less than about 75 nucleotides, less than about50 nucleotides, less than about 25 nucleotides, or less than about 15nucleotides.

In some embodiments, the 5′-end of the transferred strand has a5′-monophosphate group. In some embodiments, both, the transferredstrand and the non-transferred strand have a 5′-monophosphate group. Insome preferred embodiments, only the 5′-end of the non-transferredstrand has a 5′-monophosphate group. In some other embodiments, there isno 5′-monophosphate group on the 5′-end of the transferred strand.

The term “transposase” with respect to the present invention is intendedto mean an enzyme capable of forming a functional complex with atransposon end or transposon end sequences needed in a transpositionreaction. A transposase of the invention also includes integrases fromretrotransposons and retroviruses.

A “transposition reaction” is a reaction wherein one or more transposonends are inserted into a target DNA at random sites or almost randomsites. Essential components in a transposition reaction are atransposase and DNA oligonucleotides that exhibit the nucleotidesequences of the transposon end, including the transferred transposonend sequence and its complement, the non-transferred transposon endsequence, as well as other components needed to form a functionaltransposition complex. The method of this invention is exemplified byemploying a transposition complex formed by a hyperactive Tn5transposase and a Tn5-type transposon end (Goryshin, I. and Reznikoff,W. S., J. Biol. Chem., 273: 7367, 1998) or by a MuA transposase and a Mutransposon end comprising R1 and R2 end sequences (Mizuuchi, K., Cell,35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). However,any transposition system that is capable of inserting a transposon endin a random or in an almost random manner with sufficient efficiency to5′-tag and fragment a target DNA for its intended purpose can be used inthe present invention. Examples of transposition systems known in theart which could be evaluated for the present methods include but are notlimited to Staphylococcus aureus Tn552 (Colegio O R et al., J.Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol. Microbiol., 43:173-86, 2002), Ty1 (Devine S E, and Boeke J D., Nucleic Acids Res., 22:3765-72, 1994 and International Patent Application No. WO 95/23875),Transposon Tn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Reviewin: Curr Top Microbiol Immunol., 204: 27-48, 1996), Tn/O and IS10(Kleckner N, et al., Curr Top Microbiol Immunol., 204: 49-82, 1996),Mariner transposase (Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1(Plasterk R H, Curr Top Microbiol Immunol, 204: 125-43, 1996), P Element(Gloor, G B, Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa H,and Ohtsubo E., J Biol. Chem. 265: 18829-32, 1990), bacterial insertionsequences (Ohtsubo, F and Sekine, Y, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retroviruses (Brown P O, et al., Proc Natl Acad Sci USA,86: 2525-9, 1989), and retrotransposon of yeast (Boeke J D and Corces VG, Annu Rev Microbiol. 43: 403-34, 1989).

The method for inserting a transposon end into a target sequence can becarried out in vitro using any suitable transposon system for which asuitable in vitro transposition system is available or that can bedeveloped based on knowledge in the art. In general, a suitable in vitrotransposition system for use in the methods of the present inventionrequires, at a minimum, a transposase enzyme of sufficient purity,sufficient concentration, and sufficient in vitro transposition activityand a transposon end with which the transposase forms a functionalcomplex with the respective transposase that is capable of catalyzingthe transposition reaction. Suitable transposase transposon endsequences that can be used in the invention include but are not limitedto wild-type, derivative or mutant transposon end sequences that form acomplex with a transposase chosen from among a wild-type, derivative ormutant form of the transposase. Exemplary transposases that have beenused successfully by the Applicants in the methods of the presentinvention include wild-type or mutant forms of Tn5 transposase and MuAtransposase (although EZ-Tn5 transposase was significantly moreefficient than an equivalent protein amount of MuA transposase ingenerating 5′-tagged DNA fragments in the methods of the presentinvention), but any other transposase for which compositions andconditions for efficient in vitro transposition of defined transposonends are known or subsequently developed can be used in the presentmethods. Transposon end sequences recognized by wild-type or mutantforms of Tn5 transposase or MuA transposase are preferred, and thosetransposon end sequences that result in the highest transpositionefficiencies when complexed with the transposase, together with thecorresponding optimally active transposase enzymes that complex withthem, are most preferred for embodiments of the present invention.Preferably, a transposon is chosen wherein the transposase end sequencerequired by the transposase for transposition is not too large and thetransposon end sequences are of the minimal size possible that functionwell for the intended purpose and that are of sufficient size so thatthe same sequence is present only rarely or preferably, is not presentat all, in the target DNA or sample DNA. By way of example, thetransposon end sequences of the Tn5-derived EZ-Tn5™ transposon endsequences comprise only 19 nucleotides, whereas some other transposasesrequire much larger end sequences for transposition (e.g., MuAtransposase required transposon end sequences of approximately 51nucleotides).

Suitable in vitro transposition systems that can be used to insert atransposon end into a target nucleic acid include, but are not limitedto, those that use the EZ-Tn5™ hyperactive Tn5 Transposase availablefrom EPICENTRE Technologies, Madison, Wis., or the HyperMu™ HyperactiveMuA Transposase from EPICENTRE or another MuA Transposase, such as thatavailable from Finnzymes Oy, Espoo, Finland. Transposon endoligonucleotides that exhibit the sequences of the respective transposonends can be synthesized using an oligonucleotide synthesizer orpurchased from a commercial source based on information available fromthe respective vendors or using information well known in the art. Forexample, the nucleotide sequences of the hyperactive transposon mosaicend for EZ-Tn5™ transposase are presented in Example 1 and additionalinformation related to EZ-Tn5™ transposase is available in the publishedliterature from EPICENTRE Biotechnologies, Madison, Wis., USA.

In some embodiments, the insertion of a transposon end into target DNAaccording to the present invention can also be carried out in vivo. Iftransposition is carried out in vivo, transposition into the target DNAis preferably achieved by electroporating a synaptic complex of atransposase and a suitable transposon end composition into the host cellas described in U.S. Pat. No. 6,159,736 (herein incorporated byreference). This transposition method is exemplified by employing atransposition complex formed by a hyperactive Tn5 transposase and asuitable Tn5-type transposon end composition using methods similar tothose described by (Goryshin, I. and Reznikoff, W. S. (J. Biol. Chem.,273: 7367, 1998) or a transposition complex formed by HyperMu™Hyperactive MuA Transposase (EPICENTRE, Madison, Wis.) and a suitableMuA transposon end composition that exhibits the R1 and R2 end sequencesrecognized by the transposase. Suitable synaptic complexes or“Transposome™ complexes (EPICENTRE) between a transposon end compositionand a transposase can be made as described in U.S. Pat. No. 6,159,736and related patents of Goryshin and Reznikoff, or as described inproduct literature for Tn5-type EZ-Tn5™ Transposome™ complexes or forHyperMu™ MuA Transposome™ complexes from EPICENTRE Technologies,Madison, Wis., except that oligonucleotides that exhibit only onetransposon end are used instead of a polynucleotide or oligonucleotidethat has two transposon ends, usually at or near each end of therespective polynucleotide or oligonucleotide.

The invention also comprises kits and individual compositions for any ofthe methods of the invention. A kit is a combination of individualcompositions useful for carrying out a method of the invention, whereinthe compositions are optimized for use together in the method. Acomposition comprises an individual component or a blend of componentsfor at least one step of a method of the invention. The inventioncomprises any kit that can be assembled from a combination of any twocompositions of the invention, and any novel composition that is used ina kit or method of the invention. Alternatively, a kit may be assembledfrom a single component or composition in a convenient use format, e.g.,pre-aliquoted in single use portion, and may optionally include a set ofinstructions for use of the component or composition.

DESCRIPTION OF THE INVENTION

Introduction

The present invention relates to methods and compositions for treatingnucleic acid, and in particular, methods and compositions forfragmenting and tagging DNA using transposon compositions. The methods,compositions and kits of the present invention are useful for generatinglibraries of di-tagged linear ssDNA fragments or tagged circular ssDNAfragments (and amplification products thereof) from target DNAcomprising any dsDNA of interest (including double-stranded cDNAprepared from RNA) from any source for genomic, subgenomic,transcriptomic, or metagenomic analysis or analysis of RNA expression(e.g., for use in making labeled target for microarray analysis; e.g.,for analysis of copy number variation, for detection and analysis ofsingle nucleotide polymorphisms, and for finding genes fromenvironmental samples such as soil or water sources). The methods areuseful in a variety of processes, including, but not limited to,processes for amplification of the whole genome of one or moreorganisms, including one or more microbial or environmental organismsfor which conditions for culture or growth are unknown (e.g., wholegenome amplification or WGA), real-time PCR, emulsion PCR, comparativegenomic hybridization (CGH), comparative genomic sequencing (CGS), andfor preparing DNA-specific probes (e.g., chromosome-specific probes,e.g., chromosome paints, or e.g., gene- or locus-specific probes) forapplications such as fluorescent in situ hybridization (FISH). In someembodiments, In some embodiments, the methods are also used forgenerating templates for massively parallel DNA sequencing (so-called“next-generation sequencing”). Each of these processes or applicationsfinds uses for both research and molecular diagnostic purposes.

The present invention provides methods, compositions and kits forgenerating a library of tagged DNA fragments from target DNA comprisingdouble-stranded DNA (dsDNA) contained in any sample of interest. Themethods are easier, faster, require less hands-on time, can be performedwith smaller samples and smaller amounts of sample nucleic acids, aremore efficient in tagging both ends of the fragments, and generatedi-tagged DNA fragments that are qualitatively and/or quantitativelyrepresentative of the sample nucleic acids from which they aregenerated. The methods can be easily performed by hand without aninstrument, but also are easily adapted to robotic automation in ahigh-throughput environment.

Methods Embodiments

All of the embodiments of the methods of the present invention disclosedherein use an in vitro transposition reaction to simultaneously break atarget DNA into fragments and join a tag to the 5′-end of each fragment.Since all of the methods are related, unless otherwise specificallystated with respect to a particular embodiment, a method that is presentherein with respect to one embodiment can also be used with anotherembodiment described herein. All of the embodiments of the methodsdisclosed herein that use an in vitro transposition reaction can beperformed by assembling the reaction using either separate transposaseand transposon end compositions or a single transposome compositioncomprising a stable complex formed between the transposase and thetransposon end composition. Therefore, it will be understood that anymethod that describes the use of a transposase and a transposon endcomposition could also use a transposome composition made from thetransposase and the transposon end composition, and any method thatdescribes the use of a transposome composition could also use theseparate transposase and a transposon end compositions of which thetransposome composition is composed. This is illustrated by thefollowing two descriptions of one general method of the invention.

One embodiment of the invention is a method for generating a library oftagged DNA fragments from target DNA comprising any dsDNA of interest(e.g., for use as next-generation sequencing or amplificationtemplates), the method comprising: incubating the target DNA in an invitro transposition reaction with at least one transposase and atransposon end composition with which the transposase forms atransposition complex, the transposon end composition comprising (i) atransferred strand that exhibits a transferred transposon end sequenceand, optionally, an additional sequence 5′-of the transferred transposonend sequence, and (ii) a non-transferred strand that exhibits a sequencethat is complementary to the transferred transposon end sequence, underconditions and for sufficient time wherein multiple insertions into thetarget DNA occur, each of which results in joining of a first tagcomprising or consisting of the transferred strand to the 5′ end of anucleotide in the target DNA, thereby fragmenting the target DNA andgenerating a population of annealed 5′-tagged DNA fragments, each ofwhich has the first tag on the 5′-end; and then joining the 3′-ends ofthe 5′-tagged DNA fragments to the first tag or to a second tag, therebygenerating a library of tagged DNA fragments (e.g., comprising eithertagged circular ssDNA fragments or 5′- and 3′-tagged DNA fragments (or“di-tagged DNA fragments”)).

In one preferred embodiment, as described immediately above, the methodis performed using separate transposase and transposon end compositions,whereas in some other preferred embodiments, the method is performedusing a transposome composition comprising the complex formed betweenthe transposase and the transposon end composition.

Thus, one preferred embodiment of the invention is a method forgenerating a library of tagged DNA fragments from target DNA in an invitro transposition reaction comprising any dsDNA of interest (e.g., foruse as next-generation sequencing or amplification templates), themethod comprising: incubating the target DNA with one or moretransposome compositions, each comprising a complex between atransposase and a transposon end composition with which the transposaseforms a transposition complex, the transposon end composition comprising(i) a transferred strand that exhibits a transferred transposon endsequence and, optionally, an additional sequence 5′-of the transferredtransposon end sequence, and (ii) a non-transferred strand that exhibitsa sequence that is complementary to the transferred transposon endsequence, under conditions and for sufficient time wherein multipleinsertions into the target DNA occur, each of which results in joiningof a first tag comprising or consisting of the transferred strand to the5′ end of a nucleotide in the target DNA, thereby fragmenting the targetDNA and generating a population of annealed 5′-tagged DNA fragments,each of which has the first tag on the 5′-end; and then joining the3′-ends of the 5′-tagged DNA fragments to the first tag or to a secondtag, thereby generating a library of tagged DNA fragments (e.g.,comprising either tagged circular ssDNA fragments or 5′- and 3′-taggedDNA fragments (or “di-tagged DNA fragments”)).

In some embodiments of any of the methods of the invention, the amountof the transposase and the transposon end composition or of thetransposome composition used in the in vitro transposition reaction isbetween about 1 picomole and about 25 picomoles per 50 nanograms oftarget DNA per 50-microliter reaction. In some preferred embodiments ofany of the methods of the invention, the amount of the transposase andthe transposon end composition or of the transposome composition used inthe in vitro transposition reaction is between about 5 picomoles andabout 50 picomoles per 50 nanograms of target DNA per 50-microliterreaction. In some preferred embodiments of any of the methods of theinvention wherein the transposase is the hyperactive Tn5 transposase andthe transposon end composition comprises the MEDS transposon endcomposition or wherein the transposome composition comprises saidhyperactive Tn5 transposase and a transposon end composition thatcomprises the MEDS transposon end, the amounts of said transposase andtransposon end composition or said transposome composition used in thein vitro transposition reaction is between about 5 picomoles and about25 picomoles per 50 nanograms of target DNA per 50-microliter reaction.In some preferred embodiments of any of the methods of the inventionwherein the transposase is a hyperactive Tn5 transposase or MuAtransposase, the final concentrations of the transposase and thetransposon end composition or of the transposome composition used in thein vitro transposition reaction is at least 250 nM; in some otherembodiments, the final concentrations of hyperactive Tn5 transposase orMuA transposase and of their respective transposon end composition ortransposome composition is at least 500 nM.

In some embodiments of any of the methods of the invention, the reactiontime for the in vitro transposition reaction is two hours or less, onehour or less, 30 minutes or less, or 15 minutes or less. In somepreferred embodiments of any of the methods of the invention, thereaction time for the in vitro transposition reaction is 5 minutes orless. In some preferred embodiments of any of the methods of theinvention wherein the transposome composition comprises the hyperactiveTn5 transposase and a transposon end composition that comprises the MEDStransposon end, the reaction time for the in vitro transpositionreaction is 5 minutes or less.

In some embodiments, the method further comprises the step ofnon-selectively amplifying the tagged DNA fragments comprising di-taggedDNA fragments or tagged circular ssDNA fragments using a thermostableDNA polymerase and at least one primer that is complementary to thefirst tag or the second tag. In some preferred embodiments of the methodwhere only one transposome is use in the in vitro transpositionreaction, the step of amplifying the tagged DNA fragments comprisesamplifying the di-tagged DNA fragments or the tagged circular ssDNAfragments using a single primer that exhibits the sequence of at least aportion of the transferred strand. In some embodiments, the step ofamplifying the tagged DNA fragments using a single primer comprises aPCR or rolling circle replication reaction. In some embodiments, the 5′portion of a primer used for amplifying comprises or consists of asequencing tag domain.

In some preferred embodiment of any of the methods of the invention, thelibrary of DNA fragments is used to provide templates for DNA sequencingor nucleic acid amplification.

The invention comprises several embodiments for generating a library oftagged DNA fragments comprising either di-tagged DNA fragments or taggedcircular ssDNA fragments, as discussed below.

Use of a DNA Polymerase with Strand-Displacement or 5′ Nuclease ActivityGenerating Tagged DNA Fragments Comprising Di-Tagged DNA Fragments

One preferred embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome under conditions and for sufficient time to generate apopulation of annealed 5′-tagged DNA fragments; and then incubating thepopulation of annealed 5′-tagged DNA fragments with a DNA polymerasethat has strand-displacement or 5′ nuclease activity under conditionswithout thermocycling and wherein the annealed 5′-tagged DNA fragmentsare not denatured, wherein the DNA polymerase extends the 3′-end of eachstrand of the annealed 5′-tagged DNA fragments using the complementarystrand as a template and displaces or digests the non-transferredstrand, thereby generating the library of tagged DNA fragmentscomprising di-tagged dsDNA fragments.

One preferred embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome to generate a population of annealed_(—)5′-tagged DNAfragments; incubating the population of annealed 5′-tagged DNA fragmentswith the DNA polymerase that has strand-displacement or 5′ nucleaseactivity to generate di-tagged dsDNA fragments; and denaturing thedi-tagged dsDNA fragments to generate the library of tagged DNAfragments comprising di-tagged ssDNA fragments (e.g., by heating to 95degrees C. and rapidly cooling). In one preferred version of thisembodiment of the method, the library of tagged DNA fragments comprisingdi-tagged ssDNA fragments is generated from the target DNA in a singletube without performing any intervening purification steps.

In some embodiments of the method comprising generating a library oftagged DNA fragments comprising di-tagged DNA fragments using a DNApolymerase that has strand-displacement or 5′ nuclease activity, themethod further comprises the step of amplifying the tagged DNA fragmentscomprising di-tagged DNA fragments using a thermostable DNA polymeraseand at least one primer that is complementary to the second tag. In somepreferred embodiments of this method, the step of amplifying the libraryof tagged DNA fragments comprising di-tagged DNA fragments comprisesamplifying the library of tagged DNA fragments by PCR using only oneoligodeoxyribonucleotide that exhibits the sequence of at least aportion of the transferred strand as a PCR primer and the di-tagged DNAfragments as templates. Thus, this embodiment is a method forsingle-primer PCR amplification of a library of tagged DNA fragmentscomprising di-tagged DNA fragments generated from the target DNA. If thetarget DNA comprises total genomic DNA of an organism, this embodimentis a method for non-selective whole genome amplification.

In some preferred embodiments wherein a single transposon endcomposition is used in the in vitro transposition reaction of the methodcomprising generating the library of tagged DNA fragments comprisingdi-tagged DNA fragments using a DNA polymerase that hasstrand-displacement or 5′ nuclease activity and further amplifying thedi-tagged DNA fragments generated by PCR, two different PCR primers areused, each of which PCR primers exhibits the sequence of at least aportion of the transferred transposon end that composes the transposonend composition. In some preferred embodiments, each PCR primercomprises a 3′-portion and a 5′-portion, wherein the 3′portion exhibitsthe respective transferred transposon end sequence and the 5′-portionexhibits the sequence of a respective tag domain for a particularpurpose (e.g., a sequencing tag domain or an amplification tag domain,and optionally an address tag domain for next-generation sequencing oramplification).

In some preferred embodiments of any of the methods comprisinggenerating the library of tagged DNA fragments comprising di-tagged DNAfragments using a DNA polymerase that has strand-displacement or 5′nuclease activity, the at least one transposome in the in vitrotransposition reaction comprises or consists of two differenttransposomes. In some preferred embodiments wherein two differenttransposomes are used, each of the two transposomes comprises the sametransposase but a different transposon end composition. In somepreferred embodiments wherein two different transposomes are used, thetwo different transposomes each comprise the same transposase and thetransposon end compositions comprise different transferred strands. Insome preferred embodiments wherein two different transposomes are used,each of the two transposomes comprises different transposase enzymes anddifferent transposon end compositions, each of which forms a functionalcomplex with the respective transposase. In some preferred embodimentsof the method wherein two different transposon end compositions are usedin the in vitro transposition reaction and wherein the library of taggedDNA fragments comprising di-tagged ssDNA fragments is generated using aDNA polymerase that has strand-displacement or 5′ nuclease activity, thefirst tag exhibits the sequence of the transferred strand of onetransposon end composition and the second tag exhibits the sequence ofthe non-transferred strand of the other transposon end composition.

In some preferred embodiments of the method comprising generating thelibrary of tagged DNA fragments comprising di-tagged DNA fragments usinga DNA polymerase that has strand-displacement or 5′ nuclease activity,wherein two different transposon end compositions are used in the invitro transposition reaction, and the method further comprises the stepof amplifying the di-tagged DNA fragments generated by PCR, twodifferent PCR primers are used, one of which PCR primers exhibits thesequence of at least a portion of the transferred strand that composesone transposon end composition and the other of which PCR primersexhibits the sequence of at least a portion of the transferred strandthat composes the other transposon end composition. In some preferredembodiments, wherein the transferred strand that composes eachrespective transposon end composition comprises a 3′-portion and a5′-portion, wherein the 3′-portion exhibits the respective transferredtransposon end sequence, and the 5′-portion of each respectivetransferred strand exhibits a different the sequence comprising a tagdomain for a particular purpose (e.g., a sequencing tag domain or anamplification tag domain, and optionally an address tag domain fornext-generation sequencing or amplification), each PCR primer exhibitsthe sequence of the tag domain of the respective transferred transposonoligonucleotide.

Use of Terminal Transferase for Generating Tagged DNA FragmentsComprising Di-Tagged DNA Fragments

Another embodiment of the method comprises: incubating the target DNA inthe in vitro transposition reaction with the at least one transposome togenerate the 5′-tagged dsDNA fragments; denaturing the 5′-tagged dsDNAfragments to generate 5′-tagged ssDNA fragments; and incubating the5′-tagged ssDNA fragments with a DNA polymerase consisting of a terminaltransferase and at least one dNTP substrate for the terminal transferaseunder conditions and for sufficient time wherein the terminaltransferase joins the second tag consisting of the poly(dNMP) to the 3′end of the 5′-tagged DNA fragments, thereby generating a library oftagged DNA fragments comprising di-tagged DNA fragments (e.g., FIG. 3).In some embodiments of this method, the 3′-end of the non-transferredtransposon end oligonucleotide that composes the transposon endcomposition of the transposome is blocked (e.g., by using anon-transferred transposon end oligonucleotide that has a dideoxynucleotide or a 3′-β-methyl-nucleotide as the 3′-terminal nucleotide),which blocked 3′ nucleotide prevents addition of the poly(dNMP) by theterminal transferase, thereby preventing background tagging of thenon-transferred transposon end oligonucleotide.

Still another embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome to generate the 5′-tagged DNA fragments; incubating the5′-tagged DNA fragments, without a prior denaturation step, with a DNApolymerase consisting of a terminal transferase and at least one dNTPsubstrate for the terminal transferase under conditions and forsufficient time wherein the terminal transferase joins the second tagconsisting of the poly(dNMP) to the 3′ end of the 5′-tagged DNAfragments, thereby generating a library of tagged DNA fragmentscomprising di-tagged DNA fragments. In some embodiments of this method,the 3′-end of the non-transferred transposon end oligonucleotide thatcomposes the transposon end composition of the transposome is blocked(e.g., by using a non-transferred transposon end oligonucleotide thathas a dideoxy nucleotide or a 3′-β-methyl-nucleotide as the 3′-terminalnucleotide).

Use of a DNA Polymerase and a Terminal Tagging Oligonucleotide forGenerating Tagged DNA Fragments Comprising Di-Tagged DNA Fragments

Still another embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome to generate the 5′-tagged dsDNA fragments; denaturing the5′-tagged dsDNA fragments to generate 5′-tagged ssDNA fragments (e.g.,by heating to 95 degrees C. and rapidly cooling); and joining the secondtag to the 5′-tagged ssDNA fragments using a DNA polymerase and aterminal tagging oligonucleotide (e.g., FIG. 4), thereby generating alibrary of tagged DNA fragments comprising di-tagged DNA fragments. Insome preferred embodiments, the step of joining the second tag to the 3′end of the 5′-tagged DNA fragments using a DNA polymerase and a terminaltagging oligonucleotide comprises:

(1) Providing a terminal tagging oligonucleotide comprising orconsisting of a 5′-portion and 3′-portion, wherein the 5′-portionexhibits a sequence that is complementary to the sequence of the secondtag that it is desired to join to the 3′-termini of the 5′-tagged ssDNAfragments, and the 3′-portion exhibits a random sequence comprising orconsisting of between three and eight (e.g., 3, 4, 5, 6, 7, or 8 randomnucleotides, of which, the 3′-terminal nucleotide is blocked so that itis not capable of being extended by the DNA polymerase;

(2) contacting the 5′-tagged ssDNA fragments with the terminal taggingoligonucleotide under conditions and for sufficient time wherein theterminal tagging oligonucleotide anneals to the 5′-tagged ssDNAfragments; and

(3) contacting the 5′-tagged ssDNA fragments to which the terminaltagging oligonucleotide is annealed with the DNA polymerase in areaction mixture and under DNA polymerization conditions and forsufficient time wherein the 3′-termini of the 5′-tagged ssDNA fragmentsare extended using the terminal tagging oligonucleotide as a template,whereby the second tag is joined to their 3′-termini and 5′- and3′-tagged ssDNA fragments are generated. In some embodiments, asemi-random sequence is used in place of the random sequence in theterminal tagging oligonucleotide.

In some variants of this embodiment, the terminal taggingoligonucleotide comprises or consists of deoxyribonucleotides. In somevariants of this embodiment, the terminal tagging oligonucleotidecomprises or consists of ribonucleotides, in which embodiments the DNApolymerase is an RNA-dependent DNA polymerase. In some preferredembodiments, the 3′-portion of the terminal tagging oligonucleotideconsists of seven random nucleotides. In some preferred embodiments ofthe method wherein a terminal tagging oligonucleotide is used forjoining the second tag to the 5′-tagged ssDNA fragments, the second tagis not complementary to the first tag.

Use of a Template-Dependent (or Homologous) Ligase and a LigationTagging Oligonucleotide for Generating Tagged DNA Fragments ComprisingDi-Tagged DNA Fragments

One preferred embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome under conditions and for sufficient time to generate apopulation of annealed_(—)5′-tagged DNA fragments; and then incubatingthe population of annealed_(—)5′-tagged dsDNA fragments with atemplate-dependent (or homologous) DNA ligase and a ligation taggingoligodeoxynucleotide comprising or consisting of a 3′-portion and a5′-portion, wherein the 3′-portion exhibits a second tag that exhibitsany sequence that is desired to be joined to the 3′-end of thepopulation of annealed_(—)5′-tagged DNA fragments (e.g., an arbitrarysequence) and the 5′-portion has a 5′-monophosphate group and exhibits arandom sequence, under conditions and for sufficient time wherein thesecond tag is joined to the annealed 5′-tagged DNA fragments, therebygenerating a library of DNA fragments comprising annealed di-tagged DNAfragments. In some preferred embodiments, the method further comprisesthe step of denaturing the library of DNA fragments comprising annealeddi-tagged DNA fragments (e.g., by heating to 95 degrees C. and rapidlycooling), thereby generating a library of DNA fragments comprisingdi-tagged ssDNA fragments.

In some preferred embodiments, the ligation tagging oligonucleotidecomprises a 5′-portion that exhibits a random sequence consisting ofabout three to about eight nucleotides. In some preferred embodiments,the ligation tagging oligonucleotide comprises a 5′-portion thatexhibits a random sequence consisting of four nucleotides. In somepreferred embodiments, the template-dependent ligase is E. coli DNAligase. In one preferred version of this embodiment of the method, thelibrary of tagged DNA fragments comprising di-tagged ssDNA fragments isgenerated from the target DNA in a single tube without performing anyintervening purification steps.

Use of a Hairpin Transposon End Composition and a Template-DependentLigase for Generating a Library of Tagged DNA Fragments ComprisingTagged Circular DNA Fragments, Fantail dsDNA Fragments, or Di-Tagged DNAFragments

In one preferred embodiments of the method for generating a library oftagged DNA fragments from target DNA in an in vitro transpositionreaction, the method comprises: incubating the target DNA with one ormore transposome compositions, each comprising a complex between atransposase and a hairpin transposon end composition with which thetransposase forms a transposition complex, the hairpin transposon endcomposition comprising or consisting of a 5′-phosphate-containingoligonucleotide that exhibits a non-transferred transposon end sequenceat its 5′-end, a transferred transposon end sequence at its 3′-end, andan intervening arbitrary tag sequence between the non-transferredtransposon end sequence and the transferred transposon end sequence thatis sufficiently long to allow intramolecular stem-loop formation; underconditions and for sufficient time wherein insertion of the hairpintransposon end composition into the target DNA generates a population ofannealed 5′-tagged DNA fragments; then incubating the population ofannealed 5′-tagged DNA fragments with one or more random-sequence5′-phosphate-containing oligonucleotides which, alone, or incombination, have the same length as the single-stranded gaps in theannealed 5′-tagged DNA fragments that result following the in vitro atransposition reaction, under conditions and for sufficient time whereinthe single-stranded gaps in the population of annealed 5′-tagged DNAfragments are filled in by annealing of the random-sequenceoligonucleotides to the target DNA in the single-stranded gaps; andthen, incubating the population of annealed 5′-tagged DNA fragments withsingle-stranded gaps filled in with a template-dependent ligase underconditions and for sufficient time wherein the annealed random-sequenceoligonucleotides are ligated to each other or to the 5′-ends of adjacent5′-tagged DNA fragments, thereby generating the library of taggedcircular DNA fragments.

In another preferred embodiments of the method for generating a libraryof tagged DNA fragments from target DNA in an in vitro transpositionreaction, the method comprises: incubating the target DNA with one ormore transposome compositions, each comprising a complex between atransposase and a hairpin transposon end composition with which thetransposase forms a transposition complex, the hairpin transposon endcomposition comprising or consisting of a 5′-phosphate-containingoligonucleotide that exhibits a non-transferred transposon end sequenceat its 5′-end, a transferred transposon end sequence at its 3′-end, andan intervening arbitrary tag sequence between the non-transferredtransposon end sequence and the transferred transposon end sequence thatis sufficiently long to allow intramolecular stem-loop formation; underconditions and for sufficient time wherein insertion of the hairpintransposon end composition into the target DNA generates a population ofannealed 5′-tagged DNA fragments; then incubating the population ofannealed 5′-tagged DNA fragments with a DNA polymerase that lacks5′-to-3′ exonuclease and structure-dependent 5′ nuclease andstrand-displacement activities, under conditions and for sufficient timewherein the single-stranded gaps that are present in the population ofannealed 5′-tagged DNA fragments following the in vitro transpositionreaction are filled in by extension of the 3′ ends of each annealed5′-tagged DNA fragment by the DNA polymerase; and then, incubating thepopulation of annealed 5′-tagged DNA fragments with single-stranded gapsfilled in with a template-dependent ligase under conditions and forsufficient time wherein the 3′-ends of the annealed DNA polymeraseextension products are ligated to the 5′-ends of adjacently annealed5′-tagged DNA fragments, thereby generating the library of taggedcircular DNA fragments.

In some preferred embodiments of this method, both the DNA polymeraseand the template-dependent ligase are provided in a single reactionmixture and both the DNA polymerase extension and the template-dependentligation are carried out in the single reaction mixture.

In some embodiments of any of these methods for generating a library oftagged circular DNA fragments, the method additionally comprises, afterthe step of incubating with the template-dependent ligase to generatethe library of tagged circular DNA fragments, one or more steps toremove unligated linear ssDNA and dsDNA (e.g., comprising therandom-sequence oligonucleotides, the linear target DNA and/or thehairpin transposon end compositions that are not joined to target DNA).In one preferred embodiment for removing unligated linear ssDNA anddsDNA, the method additionally comprises: treating the reaction mixturecontaining the tagged circular DNA fragments with T5 exonuclease.

In some preferred embodiments of any of these methods for generating alibrary of tagged circular DNA fragments, the method additionallycomprises: cleaving the tagged circular DNA fragments in each of theloop structures derived from the hairpin transposon end compositions togenerate fantail dsDNA fragments, each strand of which has a portion ofthe tag on its 5′-end and a portion of the tag on its 3′-end. In someembodiments, the step of cleaving the tagged circular DNA fragments ineach of the loop structures comprises: contacting the tagged circularDNA fragments with a cleavage enzyme composition under conditions andfor sufficient time wherein the tagged circular DNA fragments arecleaved at the cleavable sites to generate the fantail dsDNA fragments.In some embodiments, the step of cleaving the tagged circular DNAfragments in each of the loop structures comprises: annealing to thetagged circular DNA fragments an oligodeoxyribonucleotide that annealsto a restriction site within the tag, and then incubating with therestriction endonuclease that cleaves at the double-stranded restrictionsite under conditions and for sufficient time to generate the library ofthe fantail dsDNA fragments. In some preferred embodiments, the step ofcleaving the tagged circular DNA fragments in each of the loopstructures comprises: contacting the tagged circular DNA fragments witha DNA glycosylase and an AP endonuclease, wherein the DNA glycosylaseremoves the nucleic acid base from a non-canonical nucleotide (e.g., adUMP or 8-oxo-dGMP) that is present within the tag and the APendonuclease cleaves the tagged circular ssDNA fragments at theresulting abasic site; in some embodiments, the DNA glycosylase isselected from among uracil-N-glycosylase and FPG protein and the APendonuclease is selected from among E. coli endonuclease III orendonuclease IV.

In some preferred embodiments of any of the methods for generating alibrary of fantail dsDNA fragments, the method additionally comprisesthe step of: denaturing the library of fantail dsDNA fragments togenerate a library of di-tagged linear ssDNA fragments.

Use of a Template-Independent Ligase for Generating Tagged DNA FragmentsComprising Tagged Circular ssDNA Fragments or Di-Tagged Linear ssDNAFragments

One preferred embodiment of the method comprises: incubating the targetDNA in the in vitro transposition reaction with the at least onetransposome, wherein the 5′-end of the transferred strand comprising thetransposome has a 5′-phosphate group, under conditions and forsufficient time to generate a population of annealed_(—)5′-tagged DNAfragments; then denaturing the annealed 5′-tagged dsDNA fragments toobtain 5′-tagged ssDNA fragments (e.g., by heating to 95 degrees C. andrapidly cooling); and then incubating the 5′-tagged ssDNA fragments in aligation reaction with a template-independent (or non-homologous) ligaseunder conditions and for sufficient time wherein the 5′-tagged ssDNAfragments are intramolecularly ligated to generate a library of taggedcircular ssDNA fragments, each of which exhibits the sequence of aportion of the target DNA and the sequence of the tag.

In one preferred version of this embodiment of the method, the libraryof tagged DNA fragments comprising tagged circular ssDNA fragments isgenerated from the target DNA in a single tube without performing anyintervening purification steps. In one preferred embodiment, thetemplate-independent ligase is selected from among bacteriophage TS2126thermostable RNA ligase and an archaeal RNA ligase (e.g.,Methanobacterium thermoautotrophicum RNA ligase 1). In some preferredembodiments, the template-dependent ligase is provided in an adenylatedform and the step of incubating the 5′-tagged ssDNA fragments with thetemplate-independent ligase is performed without adding ATP or NAD tothe ligation reaction.

In some preferred embodiments, the method further comprises: cleavingthe tagged circular ssDNA fragments at a site within the tag, therebygenerating a library of tagged DNA fragments comprising di-tagged linearssDNA fragments. In some embodiments, the step of cleaving comprisesannealing an oligodeoxyribonucleotide that is complementary to asingle-stranded restriction site within the tag of the tagged circularssDNA fragments, and then cleaving the tagged circular ssDNA fragmentsat the restriction site using the restriction endonuclease thatrecognizes the restriction site. In some other embodiments, the step ofcleaving comprises contacting the tagged circular ssDNA fragments with aDNA glycosylase and an endonuclease, wherein the DNA glycosylase removesthe nucleic acid base from a non-canonical nucleotide (e.g., a dUMP or8-oxo-dGMP) that is present within the tag and the endonuclease cleavesthe tagged circular ssDNA fragments at the resulting abasic site; insome embodiments, the DNA glycosylase is selected from amonguracil-N-glycosylase and FPG protein and the AP endonuclease is selectedfrom among E. coli endonuclease III or endonuclease IV.

In some embodiments, the method further comprises the step of amplifyingthe library of tagged DNA fragments comprising tagged circular ssDNAfragments or di-tagged linear ssDNA fragments, thereby generating anamplified library of tagged DNA fragments. In some preferredembodiments, the step of amplifying the library of tagged DNA fragmentscomprises performing a polymerase chain reaction (PCR), therebygenerating an amplified library of tagged DNA fragments comprisingamplified di-tagged DNA fragments. In some preferred embodiments, thePCR reaction is performed using a first PCR primer and a second PCRprimer, each having a 3′-portion and a 5′-portion, wherein the3′-portion of the first PCR primer is complementary to a sequenceexhibited by the tag in the tagged DNA fragments and the 3′-portion ofthe second PCR primer is complementary to a sequence that iscomplementary to the tag, and wherein each 5′ portion comprises asequencing tag domain that comprises or consists of an appropriatesequencing tag that permits use of the amplified di-tagged DNA fragmentsgenerated as templates for next-generation sequencing using a particularnext-generation sequencing platform (e.g., the Roche 454A and 454Bsequencing tags, the ILLUMINA™ SOLEXA™ sequencing tags, the AppliedBiosystems' SOLID™ sequencing tags, the Pacific Biosciences' SMRT™sequencing tags, the Pollonator Polony sequencing tags, or the CompleteGenomics sequencing tags).

Methods for Generating DNA Fragment Libraries with ImprovedRepresentation of Sequences at the Ends of the Target DNA

The inventors observed certain sequence data which indicated that therepresentation of next-generation sequence data from the ends of targetDNA comprising dsDNA molecules with a size of less than 10 Kb was lowcompared to the representation of sequence data from the middle of thattarget DNA. Without being bound by theory, one possible explanation forthis observation is that the probability of finding DNA fragments withtwo transposon end compositions inserted in opposite orientations at theends of a linear dsDNA molecule is lower than the probability of findingDNA fragments with two transposon end compositions inserted in oppositeorientations in the middle of the linear dsDNA molecule. In order tosolve this problem, the inventors developed additional methods forgenerating a DNA fragment library wherein there is a betterrepresentation of DNA fragments that exhibit the sequences at the endsof the dsDNA molecules composing the target DNA.

One preferred embodiment is a method for generating a DNA fragmentlibrary wherein there is a better representation of DNA fragments thatexhibit the sequences at the ends of the dsDNA molecules composing thetarget DNA, the method comprising: incubating the target DNA with atleast one transposome composition comprising at least one transposaseand at least one transposon end composition with which it forms atransposition complex, the transposon end composition comprising atransferred strand and a non-transferred strand, in an in vitrotransposition reaction under conditions and for sufficient time whereinthe transferred strand is joined to the target DNA, generating 5′-taggeddsDNA fragments comprising annealed 5′-tagged ssDNA fragments, each ofwhich has a first tag comprising or consisting of the transferred strandon the 5′-end; denaturing the 5′-tagged dsDNA fragments to release the5′-tagged ssDNA fragments; and then circularizing the 5′-tagged ssDNAfragments by intramolecular ligation with a template-independent ligasethat ligates ssDNA (e.g., bacteriophage TS2126 RNA ligase; e.g.,CIRCLIGASE™ thermostable ssDNA ligase, EPICENTRE, Madison, Wis., USA),thereby generating the library of tagged circular ssDNA fragments. Insome embodiments, the at least one transposase and the at least onetransposon end composition are added to the reaction as separatecomponents rather than as the single component comprising thetransposome composition. In some embodiments, the tagged circular ssDNAfragments are used as next-generation sequencing templates, or,following labeling, as target for annealing to probes on an array ormicroarray, or for other applications described elsewhere herein. Insome other embodiments, the method further comprises the step oflinearizing the tagged circular ssDNA fragments within the first tag,thereby generating di-tagged linear ssDNA fragments. In some of any ofthese embodiments comprising linearizing the tagged circular ssDNAfragments, the first tag comprises multiple tag domains, wherein thestep of linearizing the first tag results in one portion of the firsttag on the 5′ end and another portion of the first tag on the 3′-end.For example, in some embodiments, the transferred strand of thetransferred transposon end composition exhibits the first tag thatcomprises multiple tag domains (e.g., both the Roche 454A and the Roche454B sequencing tag domains), of which, at least one tag domain isjoined to the 3′ end of the di-tagged ssDNA fragments generated from thestep of linearizing the tagged circular ssDNA fragments. For example, insome embodiments, the 5′-tagged DNA fragments are generated using atransposon end composition comprising a transferred strand that containsone or more nucleotides that permit cleavage at the sites of saidnucleotides, and the step of linearizing the tagged circular ssDNAfragments within the tag comprises cleaving the tagged circular ssDNAfragments at said one or more nucleotides. For example, in someembodiments, the transferred strand contains one or more deoxyuridinenucleotides or one or more 8-oxoguanine nucleotides (e.g., synthesizedusing an oligonucleotide synthesizer), and the step of linearizing thetagged circular ssDNA fragments within the tag comprises cleaving thetagged circular ssDNA fragments by incubating the tagged circular ssDNAfragments with uracil-DNA glycosylase or formamidopyrimidine-DNAglycosylase, respectively, and an endonuclease that cleaves DNA at anabasic site (e.g., endonuclease IV). For example, in some otherembodiments, the tagged circular ssDNA fragments are linearized withinthe tag by annealing a complementary oligonucleotide to the tag andlinearizing using a restriction endonuclease that recognizes arestriction site within the double-stranded tag. In some of any of theembodiments comprising linearizing the tagged circular ssDNA fragments,the method further comprises purifying the di-tagged ssDNA fragments(e.g., using a Qiagen PCR cleanup column); in some of these embodiments,the di-tagged ssDNA fragments are used as next-generation sequencingtemplates or, following labeling, as target for annealing to probes onan array or microarray, or for other applications described elsewhereherein.

Amplification of Tagged DNA Fragments and Other Embodiments

In some embodiments of any of the methods of the invention forgenerating the library of tagged DNA fragments, the method furthercomprises: amplifying the library of tagged DNA fragments comprisingdi-tagged DNA fragments, tagged circular ssDNA fragments, or fantail DNAfragments.

In some embodiments of any of the methods, the method further comprisesstep of: amplifying the library of di-tagged linear ssDNA the taggedcircular DNA fragments or the fantail dsDNA fragments using a polymerasechain reaction (PCR). Thus, in some embodiments, the method furthercomprises (a) providing (i) first and second PCR primers, wherein atleast the 3′-end of the first PCR primer is complementary to at least aportion of the tag sequence of the tagged circular DNA fragments or toat least a portion of the tag sequence that is joined to the 3′-end ofthe fantail dsDNA fragments or to at least a portion of the tag sequencethat is joined to the 3′-end of the linear ssDNA fragments, and whereinat least the 3′-end of the second PCR primer is complementary to atleast a portion of the complement of the tag sequence of the taggedcircular DNA fragments (i.e., wherein at least the 3′-end of the secondPCR primer exhibits a sequence that is identical to at least a portionof the tag sequence), or wherein at least the 3′-end of the second PCRprimer is complementary to at least a portion of the complement of thetag sequence that is joined to the 5′-end of the fantail dsDNA fragmentsor the di-tagged linear ssDNA fragments (i.e., wherein at least the3′-end of the second PCR primer exhibits a sequence that is identical toat least a portion of the tag sequence that is joined to the 5′-end ofthe fantail dsDNA fragments or the di-tagged linear ssDNA fragments),and (ii) a thermostable DNA polymerase that can be used for PCR; and (b)incubating the tagged circular DNA fragments or the fantail dsDNAfragments or the di-tagged linear ssDNA fragments with the respectivefirst and the second PCR primers and the thermostable DNA polymeraseunder PCR amplification conditions and for sufficient time whereinamplified di-tagged linear dsDNA fragments are generated.

In some embodiments wherein the method comprises amplifying the taggedcircular DNA fragments using PCR, the first PCR primer is complementaryto a tag sequence in at least a portion of the loop structure of thehairpin transposon end composition that is inserted into the target DNAof the tagged circular DNA fragments and/or the second PCR primerexhibits a sequence that is identical to at least a portion of the loopstructure of the hairpin transposon end composition that is insertedinto the target DNA of the tagged circular DNA fragments. In someembodiments, the first PCR primer is complementary to at least a portionof the transferred transposon end sequence or the non-transferredtransposon end sequence and the second PCR primer is identical to atleast a portion of the transferred transposon end sequence or thenon-transferred transposon end sequence.

In some embodiments, the 5′ portion of the first PCR primer or the 5′portion of the second PCR primer, or the 5′ portions of both the firstand the second PCR primers comprise or consist of first or secondsequencing tags, respectively, for generation of templates fornext-generation sequencing for a particular sequencing platform (e.g.,sequencing tags for: a ROCHE 454A or 454B sequencing platform; for anILLUMINA SOLEXA sequencing platform; for an APPLIED BIOSYSTEMS SOLID™sequencing platform; for a PACIFIC BIOSCIENCES' SMRT™ sequencingplatform; for a POLLONATOR POLONY sequencing platform; for a HELICOSsequencing platform; for a COMPLETE GENOMICS sequencing platform; for anINTELLIGENT BIOSYSTEMS sequencing platform; or for any other sequencingplatform). In some embodiments, the 5′ portion of the first PCR primeror the 5′ portion of the second PCR primer additionally comprises orconsists of an address tag domain or another tag domain for a particularpurpose. In other embodiments, the tag of the tagged circular DNAfragments comprises a sequencing tag for next-generation sequencingusing a particular platform.

In embodiments of the method wherein a library of tagged DNA fragmentscomprising di-tagged DNA fragments is generated using a DNA polymerasethat has 5′ nuclease or strand-displacement activity, the step ofamplifying the library comprises using only a singleoligodeoxyribonucleotide primer that is complementary to the second tagto amplify the library of tagged DNA fragments by PCR. In someembodiments, the single primer used for PCR exhibits at least a portionof the transferred transposon end sequence. In some other embodiments,the single primer used for PCR exhibits at least a portion of thesequence of the 5′-portion of the transferred transposon endoligonucleotide. In some other preferred embodiments, the step ofamplifying the library of tagged DNA fragments comprising di-tagged DNAfragments using a single oligonucleotide primer comprises: providing asingle oligonucleotide primer that is complementary to the second tag atthe 3′ end of the tagged DNA fragments and a thermostable DNA polymerasethat is suitable for PCR; and incubating the library of tagged DNAfragments with the oligonucleotide primer and the thermostable DNApolymerase under PCR amplification conditions for sufficient timewherein the library of tagged DNA fragments is PCR amplified, generatinga library of amplified tagged DNA fragments.

In some other embodiments wherein a library of tagged DNA fragmentscomprising di-tagged DNA fragments is not generated using a DNApolymerase that has 5′ nuclease or strand-displacement activity, thestep of amplifying the library comprises performing a polymerase chainreaction (PCR), the method further comprising: (1) providing (a) firstand second PCR primers, wherein at least the 3′-end of the first PCRprimer is complementary to at least a portion of the first tag at the 3′end of the di-tagged DNA fragments or the fantail DNA fragments or to atleast a portion of the tag in the tagged circular ssDNA fragments and atleast the 3′-end of the second PCR primer is complementary to at least aportion of the complement of the second tag of the di-tagged DNAfragments or the fantail DNA fragments or to at least a portion of thecomplement of the tag in the tagged circular ssDNA fragments, and (b) athermostable DNA polymerase that is suitable for PCR; and (2) incubatingthe library of tagged DNA fragments with the PCR primers and thethermostable DNA polymerase under PCR amplification conditions and forsufficient time wherein the library of tagged DNA fragments is amplifiedto generate a library of amplified tagged DNA fragments. In someembodiments, the first or the second PCR primer comprises a 5′ portionand a 3′ portion, wherein the 5′ portion is not complementary to thesequence in the respective tag or its complement in the tagged DNAfragments and the 3′ portion is complementary to the sequence of therespective tag or its complement. In some embodiments, the 5′ portion ofthe first and second PCR primers comprise or consist of the appropriatefirst and second sequencing tags that permit their use to generatetemplates for next-generation sequencing (e.g., the Roche 454A and 454Bsequencing tags or the appropriate first and second sequencing tags foranother sequencing platform; e.g., without limitation, the IlluminaSolexa or the Applied Biosystems Solid platform).

A wide variety of enzymes and kits are available for performing theamplification reaction by PCR. For example, in some embodiments, the PCRamplification is performed using either the FAILSAFE™ PCR System or theMASTERAMP™ Extra-Long PCR System from EPICENTRE Biotechnologies,Madison, Wis., as described by the manufacturer. These systems permitrapid optimization of the PCR reaction conditions using a series of2×PCR PreMixes provided with each system to identify the optimal PreMixfor a particular template and primer pair. However, the invention is notlimited to the use of those products or conditions for the amplificationreaction and any suitable thermostable DNA polymerase and reactionmixture that permits amplification of the sequence between the primerthat anneals to the target sequence and the primer that anneals to thetransposon can be used.

The invention is also not limited to the use of PCR to amplify thelibrary of tagged DNA fragments. Any suitable amplification method(e.g., rolling circle amplification, riboprimer amplification (e.g.,U.S. Pat. No. 7,413,857), ICAN, UCAN, ribospia, terminal tagging (U.S.Patent Application No. 20050153333), Eberwine-type aRNA amplification orstrand-displacement amplification) that amplifies the same sequence, andgenerates a suitable composition and amount of amplification product forthe intended purpose can be used in embodiments of the presentinvention. For example, some strand displacement methods that can beused are described in PCT Patent Publication Nos. WO 02/16639; WO00/56877; and AU 00/29742; of Takara Shuzo Company, Kyoto, Japan; U.S.Pat. Nos. 5,523,204; 5,536,649; 5,624,825; 5,631,147; 5,648,211;5,733,752; 5,744,311; 5,756,702; and 5,916,779 of Becton Dickinson andCompany; U.S. Pat. Nos. 6,238,868; 6,309,833; and 6,326,173 ofNanogen/Becton Dickinson Partnership; U.S. Pat. Nos. 5,849,547;5,874,260; and 6,218,151 of Bio Merieux; U.S. Pat. Nos. 5,786,183;6,087,133; and 6,214,587 of Gen-Probe, Inc.; U.S. Pat. No. 6,063,604 ofWick et al.; U.S. Pat. No. 6,251,639 of Kurn; U.S. Pat. No. 6,410,278;and PCT Publication No. WO 00/28082 of Eiken Kagaku Kabushiki Kaishi,Tokyo, Japan; U.S. Pat. Nos. 5,591,609; 5,614,389; 5,773,733; 5,834,202;and 6,448,017 of Auerbach; and U.S. Pat. Nos. 6,124,120; and 6,280,949of Lizardi.

In preferred embodiments of the invention, is not necessary to sizeselect the library of 5′-tagged DNA fragments generated in the in vitrotransposition reaction or the final library of tagged DNA fragments. Inthe event size selection or purification is necessary for certainapplications, the 5′-tagged DNA fragments can be size selected byagarose gel electrophoresis (e.g., using a low-melting-temperaturenon-denaturing agarose gel of an appropriate percentage agarose for thedesired size range of DNA fragments), and purified (e.g., to remove theun-inserted transposon end oligonucleotides, other reaction products,and agarose gel; e.g., by digestion of the portion of the agarose gelcontaining the desired size range of 5′-tagged DNA fragments withGELase™ agarose gel-digesting enzyme, EPICENTRE Biotechnologies,Madison, Wis., USA, followed by alcohol precipitation, and otherclean-up steps according to directions with the GELase product, or usingany other purification method known in the art). In some embodiments, apurification step comprising polyethylene glycol (PEG) precipitation isused to precipitate the library of tagged DNA fragments withoutprecipitating contaminating substances (e.g., without limitation,unligated ligation tagging oligonucleotides or other reactioncomponents). In some embodiments, a spin column or any otherpurification method known in the art is used.

In some embodiments, the tagged circular DNA fragments are used astemplates for DNA sequencing.

In some embodiments, the tagged DNA fragments are used as templates forDNA sequencing.

In some embodiments, the library of tagged DNA fragments is used astemplate for an amplification reaction (e.g., a PCR amplificationreaction using PCR primers that are complementary to the first and thesecond tags of tagged DNA fragments comprising di-tagged DNA fragmentsor fantail DNA fragments or that are complementary to the tag of taggedDNA fragments comprising tagged circular ssDNA fragments). In somepreferred embodiments, the library of amplified tagged DNA fragmentscomprises most or approximately all of the sequences exhibited by thetarget DNA. In some embodiments wherein the target DNA comprises genomicDNA of an organism, the amplification reaction is a whole genomeamplification reaction.

In some embodiments of the method comprising amplifying the tagged DNAfragments, the amplified are labeled by incorporation of a labelednucleotide during one or more steps of the amplification method (e.g.,the PCR amplification reaction method). In some embodiments, the libraryof amplified tagged DNA fragments that contain the label is used todetect or capture or to detect and capture the amplified tagged DNAfragments that contain the label for a particular application.

Some embodiments of any of the methods of the invention for generating alibrary of tagged DNA fragments (e.g. di-tagged DNA fragments) comprisegenerating a library of “labeled” tagged DNA fragments that contain oneor multiple moieties (e.g., one or multiple affinity-binding molecules)that permit capture of the labeled tagged DNA fragments on a surface, orone or multiple detectable moieties that permit detection of the labeledtagged DNA fragments (e.g., which anneal to a complementary DNA, such ascomplementary DNA in a chromosome). Also, some embodiments of any of themethods of the invention comprising further amplifying the library oftagged DNA fragments comprise generating a library of “labeled”amplified tagged DNA fragments comprising one or multiple moieties(e.g., one or multiple affinity-binding molecules) that permit captureon a surface, or one or multiple detectable moieties that permitdetection of the labeled tagged DNA fragments (e.g., which anneal to acomplementary DNA, such as complementary DNA in a chromosome). In someembodiments, the library of labeled tagged DNA fragments or labeledamplified tagged DNA fragments is generated by using at least onelabeled oligonucleotide (e.g., a labeled transferred transposon endoligonucleotide, a labeled ligation tagging oligonucleotide, or at leastone labeled amplification primer, such as at least one (or more thanone) PCR primer). In some other embodiments, a library of labeledamplified tagged DNA fragments is generated by including a labeled dNTPthat is incorporated into the amplification products during theamplification reaction. The labeled dNTP can have any label known in theart that can be used for generating labeled amplified tagged DNAfragments, whether by direct labeling or by indirect labeling. By“direct labeling”, we mean that the capture moiety or detectable labelis attached directly to the amplified tagged DNA fragments without anyother moiety between the capture or detectable moiety and the tagged DNAfragment or amplified tagged DNA fragment. By “indirect labeling”, wemean that there is at least one other moiety between the capture ordetectable moiety and the tagged DNA fragment or amplified tagged DNAfragment. One example of direct labeling is incorporating a dye-labelednucleotide into the tagged DNA fragments, whereas one example ofindirect labeling is incorporating a biotin-labeled nucleotide into thetagged DNA fragments and then labeling the tagged DNA fragments with adye detectable moiety by incubating with dye-labeled streptavidin underconditions wherein the dye-labeled streptavidin binds to thebiotin-labeled nucleotides. The invention comprises use of any suitablemethod for generating the library of labeled tagged DNA fragments orlabeled amplified tagged DNA fragments, wherein the label issubsequently used for capture or detection.

In some other embodiments, tagged DNA fragments in a library preparedusing a method of the invention are subsequently labeled, directly orindirectly, by contacting the library of tagged DNA fragments with areactive dye molecule (e.g., any of the reactive fluorescent dyescontaining an N-hydroxysuccinimidyl or “NHS” ester from MolecularProbes, Eugene, Oreg.) or with a reactive affinity-binding molecule(e.g., a reactive biotinylation reagent, such as a biotin-NHS compound,from Pierce Chemical Company, Rockford, Ill.). For example, in someembodiments, the library of labeled amplified tagged DNA fragments isgenerated by incorporating a dNTP that contains an aminoallyl-groupduring the amplification reaction, and then the library of amplifiedtagged DNA fragments containing the aminoallyl-group is contacted withthe labeled fluorescent dye-NHS ester or the biotin-NHS ester togenerate a fluorescent dye-labeled amplified tagged DNA fragments orbiotin-labeled amplified tagged DNA fragments, respectively. Those withknowledge in the art will know or know how to find many additionalspecific methods and reagents, including kits, e.g., from MolecularProbes, for labeling the library of amplified tagged DNA fragments for aparticular purpose (e.g. to permit capture on a surface or detection).For example, Examples include one or more modified nucleotides that hasan aminoallyl-group, a propynyl-group, a biotin group, a fluorescent orother detectable dye, or any other detectable molecule or combination ofmolecules known in the art, including quantum dots, an enzyme (e.g., aphosphatase, a peroxidase, or a pyrophosphatase), or a detectableprotein (e.g., phycobiliprotien, phycoerythrin). In some otherembodiments, a library of labeled amplified tagged DNA fragments isgenerated by incorporation of one or more modified dNTPs that arelabeled with an affinity-binding molecule or a detectable moiety duringthe amplification reaction, e.g., during a PCR amplification reaction,e.g., by incorporation of one or more modified dNTPs that has anaminoallyl-group, a biotin group, a fluorescent or other detectable dye,or another moiety that permits it to be detected, either directly, orindirectly following labeling with any other detectable molecule orcombination of molecules known in the art, including quantum dots, or anenzyme or detectable protein (e.g., phycobiliprotein, phycoerythrin)that is linked to an affinity binding molecule (e.g., as streptavidin,an antibody).

In some embodiments, the tagged DNA fragments (e.g., di-tagged DNAfragments are used for preparation of labeled DNA fragments forhybridization to probes attached to a surface (e.g., as labeled targetDNA for hybridization to DNA probes on an array or microarray). In someembodiments, tagged DNA fragments (e.g., comprising di-tagged DNAfragments) are used for hybridization to chromosomes or parts ofchromosomes in fixed cells or tissue sections (e.g., for fluorescent insitu hybridization or FISH).

In some embodiments, the method comprises generating labeled tagged DNAfragments or labeled amplified tagged DNA fragments (e.g., labeleddi-tagged DNA fragments or labeled amplified di-tagged DNA fragments)for use in hybridization to chromosomes (e.g., wherein the labeledtagged DNA fragments are prepared from target DNA comprising DNA fromone or more specific chromosomes for use as “chromosome paints” (e.g.,for hybridization to one or more chromosomes in fixed cells or tissuesections, e.g., using fluorescent in situ hybridization or FISH forapplications such as typing chromosomes, or for research, medicaldiagnostics, identifying the sex of an organism, or other cellbiological applications). In some embodiments, the method comprisesgenerating labeled tagged DNA fragments or labeled amplified tagged DNAfragments from target DNA comprising parts of chromosomes (e.g., whereinthe tagged DNA fragments are prepared from DNA encoding one or morespecific genes or loci of one or more chromosomes (e.g., forhybridization to one or more chromosomes in fixed cells or tissuesections, e.g., using fluorescent in situ hybridization or FISH, or foruse as gene-specific or loci-specific probes in in vitro assays forapplications such as analyte-specific assays or diagnostic tests formedical, industrial, environmental, or molecular or cell biologyresearch applications).

In some embodiments, hybridization of labeled tagged DNA fragments toprobes on a surface (e.g., an array or microarray, a dipstick, a quantumdot, a bead, or a microchannel in a microfluidic device) is used fordetecting, quantifying, determining relative quantities, orcharacterizing one or more DNA molecules or portions thereof that is inor from a natural source (e.g. genomic DNA from a cell; e.g., human DNAfor evaluation of copy-number variation or “CNV”, or DNA from apathogenic bacterial, fungal, mycoplasmal, viral, or nematode cell thatis a pathogen), or from an in vitro source (e.g., double-stranded cDNAmade by reverse transcription of RNA, such as mRNA or non-coding RNA orviral RNA, that is isolated from a natural source or that is amplifiedfrom a natural source using a nucleic acid amplification method, such asa DNA or RNA amplification method).

In some other embodiments wherein the method comprises amplifying thetagged DNA fragments, the method comprises generating labeled amplifiedtagged DNA fragments by incorporating one or more modified dNTPs thathas an affinity-binding molecule or a detectable moiety during theamplification reaction, e.g., during a PCR amplification reaction (e.g.,by incorporation of one or more modified dNTPs that has anaminoallyl-group, a biotin group, a fluorescent or other detectable dye,or another moiety that permits it to be detected, either directly, orindirectly following labeling with any other detectable molecule orcombination of molecules known in the art, including quantum dots, or anenzyme or detectable protein (e.g., phycobiliprotein, phycoerythrin)that is linked to an affinity binding molecule (e.g., as streptavidin,an antibody).

In some other embodiments, the tagged DNA fragments or amplified taggedDNA fragments prepared using a method of the invention are labeled byincorporation of one or more modified dNTPs that has an affinity-bindingmolecule or a detectable moiety during the amplification reaction (e.g.,during the respective transcription, RCR or PCR reaction, e.g., byincorporation of one or more modified dNTPs that has anaminoallyl-group, a biotin group, a digoxigenin group, a fluorescent orother detectable dye, or another moiety that permits it to be detected,either directly, or indirectly following labeling with any otherdetectable molecule or combination of molecules known in the art,including quantum dots, or an enzyme or detectable protein (e.g.,phycobiliprotein, phycoerythrin) that is linked to an affinity bindingmolecule (e.g., as streptavidin, an antibody). In some embodiments, therespective products are used for preparation of labeled nucleic acidfragments for hybridization to probes attached to a surface (e.g., aslabeled target nucleic acid for hybridization to DNA probes on an arrayor microarray). In some embodiments, the respective labeled products areused for hybridization to chromosomes or parts of chromosomes in fixedcells or tissue sections (e.g., for fluorescent in situ hybridization orFISH). In some embodiments, hybridization of labeled products to probeson a surface is used for detecting, quantifying, determining relativequantities, or characterizing one or more portions of a target DNA froma natural source (e.g. genomic DNA from a cell; e.g., for evaluation ofcopy-number variation or “CNV”) or from an in vitro source (e.g.,double-stranded cDNA made by reverse transcription of RNA, such as mRNAor non-coding RNA (ncRNA), that is isolated from a natural source orthat is amplified from a natural source using an RNA amplificationmethod).

In some embodiments of methods comprising generating a library of taggedcircular DNA fragments, the transferred transposon end oligonucleotide,in addition to exhibiting the sequence of the transferred transposon endin its 3′ portion, also exhibits a sequence of one strand of adouble-stranded RNA polymerase promoter in its 5′ portion. In someembodiments of methods comprising generating a library of di-tagged DNAfragments using a ligation tagging oligonucleotide and atemplate-dependent ligase, the ligation tagging oligonucleotide exhibitsa sequence of one strand of a double-stranded RNA polymerase promoter inits 3′ portion. In some embodiments of methods wherein the transferredtransposon end oligonucleotide or the ligation tagging oligonucleotidedoes not exhibit an RNA polymerase promoter sequence, the method furthercomprises PCR amplifying the di-tagged DNA fragments using at least onePCR primer that is a “promoter primer.” The promoter primer has a“5′-flap” or “5′-tail” portion that does not anneal to the di-tagged DNAfragments and that exhibits the sequence of one strand of adouble-stranded RNA polymerase promoter, and a 3′ portion that annealsto the first or second tag of the 5′- and 3′-tagged DNA fragments ortheir complements.

In some preferred embodiments wherein the transferred transposon endoligonucleotide, the ligation tagging oligonucleotide, or a PCR primerexhibits an RNA polymerase promoter sequence, the RNA polymerasepromoter is a T7-type RNA polymerase promoter and the method furthercomprises the step of transcribing the 5′- and 3′-tagged DNA fragmentsin vitro using a T7-type RNA polymerase that recognizes the promoter.Most preferably, the RNA polymerase and promoter are chosen from amongT7 RNAP, T3 RNAP and SP6 RNAP and the corresponding cognate promoters.However, transcription steps of a method of the invention can use anyRNAP for which a suitable promoter sequence that permits transcriptionwith high specificity is known or can be obtained. Kits and enzymes forin vitro transcription are commercially available from many vendors andthe appropriate reaction mixtures and conditions for carrying out stepsof the present invention comprising in vitro transcription can use thoseproducts as described by the manufacturers. For example, in vitrotranscription using T7 RNAP can be carried out using the AMPLISCRIBE™T7-Flash™ Transcription Kit or the AMPLISCRIBE™ T7 High YieldTranscription Kit from EPICENTRE Biotechnologies, Madison, Wis. asdescribed in the product literature. Similarly, if T3 RNAP or SP6 RNAPis used in a method of the invention for in vitro transcription, anAMPLISCRIBE™ T3-Flash™ High Yield Transcription Kit or with theAMPLISCRIBE™ SP6 High Yield Transcription Kit (EPICENTREBiotechnologies, Madison, Wis.), respectively, can be used as described.

In some embodiments, the transferred transposon end oligonucleotide, theligation tagging oligonucleotide, or a PCR primer exhibits, in additionto the RNA polymerase promoter sequence, additional sequences fortranslation, such as but not limited to a ribosome binding site and atranslation start codon (also referred to as a “translation startsignal”), and the method additionally comprises translating thetranscribed RNA. In some of these embodiments, the method furthercomprises the step in vitro translation of the resulting RNAtranscripts. Systems and kits for in vitro translation of the RNAtranscripts are also commercially available from many sources and can beused for the present invention. By way of example but not of limitation,rabbit reticulocyte lysate, wheat germ extract, and E. coli S30 extractsystems from PROMEGA Corporation, Madison, Wis. can be used for thepresent invention. Still further, kits for coupled in vitrotranscription and in vitro translation are also commercially availableand can be used, such as TNT® Quick Coupled Transcription/TranslationSystems from Promega.

In some preferred embodiments of the method, the library of di-taggedDNA fragments generated from target DNA comprising DNA sample from awhole genome of a cell or organism are PCR amplified (i.e., the methodcomprises or consists of a method for whole genome amplification). Insome embodiments, the method for whole genome amplification is used toamplify a whole genome from a single cell. In some preferred embodimentsof the whole genome amplification method herein, the library of taggedDNA fragments is generated from a DNA sample from a whole genome of acell or organism are PCR amplified using the single oligonucleotideprimer (or PCR primer) that is complementary to the second tag.

In some embodiments, the tagged DNA fragments generated using a methodof the invention are generated from target DNA comprising or consistingof genomes and/or double-stranded cDNA prepared from RNA from allorganisms (e.g., multiple organisms) that are present in anenvironmental sample (e.g., for metagenomic or metatranscriptomicapplications, including for industrial, medical, or researchapplications).

In some other embodiments of the method, the library of tagged DNAfragments is generated from target DNA comprising DNA comprising orconsisting of a single chromosome or a portion of a chromosome. In someof these embodiments, the method comprises PCR amplifying library oftagged DNA fragments generated from the target DNA comprising orconsisting of DNA of a single chromosome or a portion of a chromosome,including a portion of a chromosome comprising one or more genes or geneloci under conditions wherein the PCR-amplified products are labeledwith a detectable moiety (e.g., a fluorescent, infrared-fluorescent,chemiluminescent, visible, or other detectable dye; e.g., using adye-labeled dNTP in the PCR. In some embodiments, the PCR-amplifiedproducts that are labeled with the detectable moiety are used forstaining fixed cells in situ (e.g., the PCR amplification products areused as chromosome paints). Thus, in some preferred embodiments, themethod comprises or consists of a method for making chromosome paints orsub-chromosome paints or chromosome markers.

In some embodiments, the tagged DNA fragments or the amplified taggedDNA fragments generated using the method are used as the target DNA fora second round of fragmentation and tagging using a method of theinvention. In some embodiments, the same transposome is used in both thefirst and second rounds of the method. In some embodiments, a seconddifferent transposase and different transposon ends are used for thesecond round.

In some embodiments, the tagged DNA fragments or the amplified taggedDNA fragments generated using the method are cloned in a vector (e.g.,in a COPYCONTROL™ fosmid vector, EPICENTRE Biotechnologies, Madison,Wis., USA). In some embodiments wherein the method further comprisescloning the tagged DNA fragments or the amplified tagged DNA fragmentsand wherein the tagged DNA fragments or the amplified tagged DNAfragments (e.g., PCR-amplified tagged DNA fragments exhibits an RNApolymerase promoter, the method further comprises transcribing at leastone strand of the cloned tagged DNA fragments or the amplified taggedDNA fragments. In some embodiments, the cloned tagged DNA fragments orthe amplified tagged DNA fragments are transcribed in vitro using an RNApolymerase that recognizes the RNA polymerase promoter. In someembodiments, the cloned tagged DNA fragments or the amplified tagged DNAfragments are transcribed in vivo in a host cell that is capable ofinducible expression of the RNA polymerase that recognizes the RNApolymerase promoter and then transcribing DNA templates that contain thepromoter to which the RNA polymerase binds (e.g., the pET system iswidely used for expression of proteins in vivo from an induced T7-typeRNA polymerase). In some preferred embodiments, the RNA polymerase forin vitro or in vivo expression is a T7-type RNA polymerase andtranscription is initiated from a respective cognate T7-type RNAPpromoter. In some preferred embodiments, the T7-type RNA polymerase isselected from among T7 RNA polymerase, T3 RNA polymerase, and SP6 RNApolymerase.

In some embodiments of any of the methods, either the transferredtransposon end oligonucleotide, the ligation tagging oligonucleotide, ora PCR primer, contains or is joined to an affinity molecule (e.g.,biotin or digoxigenin), and the method additionally comprises the stepsof: providing a solid surface that is covalently or non-covalentlycoated with an affinity binding substance that is capable ofspecifically binding and forming a specific binding pair with theaffinity molecule (e.g., streptavidin or avidin for binding biotin, oran antibody for binding digoxigenin); and, either prior to or followingthe step in which it is involved, contacting the products generatedusing the transferred transposon end oligonucleotide, the ligationtagging oligonucleotide, or the PCR primer that is chemically joined tothe affinity molecule under conditions and for sufficient time whereinit binds to affinity binding substance that is joined to the solidsurface.

The invention is not limited to a particular solid surface, which can beporous or non-porous, and of any composition, size or shape that issuitable for the particular method and application. By way of example,but not of limitation, the solid surface can be selected from the groupconsisting of: magnetic beads, coated beads, slides, the wells of amicrotiter plate, tubes, and dipsticks consisting of glass, plastic(e.g., latex or polystyrene), silica, Teflon, or another suitablematerial. The purpose of the solid surface that is coated with theaffinity binding substance is to permit manipulation (e.g., capture andwashing to remove from other molecules in a reaction mixture),isolation, and capture of the transferred transposon endoligonucleotide, the ligation tagging oligonucleotide, or the PCR primerthat is chemically joined to the affinity molecule, or to permitmanipulation, isolation, and capture of the 5′-tagged DNA fragments, the5′- and 3′-tagged DNA fragments, or the PCR products generatedtherefrom. In order to prevent non-specific binding, in someembodiments, the solid support is treated with a large excess of asubstance selected from the group consisting of: DNA-free tRNA; protein(e.g. BSA), polysaccharide (e.g., glycogen, dextran sulphate, orheparin). The invention is also not limited to a specific affinitymolecule or affinity binding substance, so long as they are capable ofspecifically binding and forming a specific binding pair.

Thus, in some embodiments, the tagged DNA fragments or the amplifiedtagged DNA fragments are captured, isolated, purified, or used inanother method by binding to the solid surface, the method comprisingthe steps of: contacting the tagged DNA fragments or the amplifiedtagged DNA fragments that contains the affinity molecule with the solidsurface in the presence of reagents and under conditions that facilitateits binding to the affinity-binding substance that is attached to thesolid surface, wherein the tagged DNA fragments or the amplified taggedDNA fragments are bound to the surface.

In some preferred embodiments, the affinity molecule is biotin and theaffinity binding substance is avidin or streptavidin, or wherein theaffinity molecule is digoxigenin and the affinity binding substance isan antibody that specifically binds digoxigenin.

As used herein, the terms “transposase” and “DNA polymerase” and“ligase” refer to protein molecules or protein molecule aggregates thatare responsible for catalyzing specific chemical and biologicalreactions. In general, a method, composition, or kit of the invention isnot limited to use of a particular transposase or DNA polymerase enzymefrom a particular source. Rather, a method, composition, or kit of thepresent invention comprises any transposase or DNA polymerase enzymefrom any source that has an equivalent enzymatic activity to theparticular enzymes disclosed herein with respect to the particularmethod, composition, or kit. Still further, the methods of the presentinvention also include embodiments wherein any one particular enzymethat is provided and used in a step of the method is replaced by acombination of two or more enzymes which, when used in combination,whether used separately in a stepwise manner or used together at thesame time reaction mixture, result in results that are identical to theresults obtained using the one particular enzyme. The methods, buffers,and reaction conditions presented herein, including in the examples, arepresently preferred for the embodiments of the methods, compositions,and kits of the present invention. However, other enzyme storagebuffers, reaction buffers, and reaction conditions for use of some ofthe enzymes of the invention are known in the art, which may also besuitable for use in the present invention, and are included herein.

Composition and Kit Embodiments

The invention also comprises kits and compositions for a method of theinvention. A kit is a combination of individual compositions useful forcarrying out a method of the invention, wherein the compositions areoptimized for use together in the method. A composition comprises anindividual component for at least one step of a method of the invention.The invention comprises any kit that can be assembled from a combinationof any two novel compositions or kits of the invention, or from anynovel composition that is used in a kit. In some embodiments, the kit orcomposition comprises or consists of a subset of any kit or compositiondescribed here, in any appropriate combination and for any reason, suchas to provide the user flexibility to adapt the method for a particularpurpose or application, or to permit the user to employ othercompositions together with the kit or composition comprising orconsisting of the subset.

Composition Embodiments

One embodiment of a composition of the invention is a transposomecomposition comprising (i) a transferred strand that has a 3′-portionthat exhibits the transferred transposon end sequence and a 5′-portionthat exhibits the sequence of a tag domain, and (ii) a5′-phosphate-containing non-transferred strand that exhibits only thenon-transferred transposon end sequence, wherein the transposase forms acomplex with the transposon end composition that is active in an invitro transposition reaction. In some embodiments, the tag domain is atag domain for use in next-generation sequencing or amplification. Insome embodiments, the tag domain is selected from among a restrictionsite domain, a capture tag domain, a sequencing tag domain, a detectiontag domain, an address tag domain, an amplification tag domain, and atranscription promoter domain.

One other composition of the invention is a transferred transposon endcomposition wherein the transferred strand comprises a 3′-portion and a5′-portion, wherein the 3′-portion exhibits a transferred transposon endsequence and the 5′-portion comprises a transcription promoter domainthat exhibits an RNA polymerase promoter sequence.

Another composition of the invention is a hairpin transposon endcomposition comprising or consisting of a 5′-phosphate-containingoligonucleotide that exhibits a non-transferred transposon end sequenceat its 5′-end, a transferred transposon end sequence at its 3′-end, andan intervening arbitrary tag sequence between the non-transferredtransposon end sequence and the transferred transposon end sequence thatis sufficiently long to allow intramolecular stem-loop formation. Insome preferred embodiments, the hairpin transposon end compositioncomprises exhibits the transposon end sequences of the hyperactive Tn 5transposase. In some other embodiments, the hairpin transposon endcomposition is adenylated on its 5′-end rather than having a5′-phosphate group.

The invention also comprises compositions for performing the methods.For example, one composition of the invention is an oligonucleotidecomprising a 3′-portion and a 5′-portion, wherein the 3′-portionexhibits a transferred transposon end sequence and the 5′-portionexhibits a restriction site domain (e.g. for a rare-cutting restrictionendonuclease such as NotI or AscI, or for a type II restrictionendonuclease such as FokI). For example, one other composition of theinvention is an oligonucleotide comprising a 3′-portion and a5′-portion, wherein the 3′-portion exhibits a transferred transposon endsequence and the 5′-portion exhibits an RNA polymerase promoter sequence(e.g., for phage T7, T3, SP6 or N4 RNA polymerase). In some preferredembodiments, the RNA polymerase promoter sequence is a sense promotersequence for any of these RNA polymerases. In some other embodiments,the RNA polymerase promoter sequence is an anti-sense promoter sequencefor any of these RNA polymerases. One other composition of the inventionis an oligonucleotide comprising a 3′-portion and a 5′-portion, whereinthe 3′-portion exhibits a transferred transposon end sequence and the5′-portion exhibits a tag domain selected from among a sequencing tagdomain, an amplification tag domain, a capture tag domain, an addresstag domain, a detection tag domain, and a restriction site tag domain.

In some preferred embodiments, the transferred transposon end sequenceis the MEDS or pMEDS transferred transposon end composition for EZ-Tn5™transposase (EPICENTRE). In some preferred embodiments, the sequence tagdomain exhibits a sequencing tag that is appropriate for a ROCHE 454sequencing platform, an ILLUMINA™ SOLEXA™ sequencing platform, a LIFETECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™ sequencing platform, a PACIFICBIOSCIENCES' SMRT™ sequencing platform, a POLLONATOR Polony sequencingplatform, a COMPLETE GENOMICS sequencing platform, an INTELLIGENTBIOSYSTEMS' sequencing platform, or a HELICOS sequencing platform.

Kit Embodiments

One embodiment of the invention is a kit for generating a library of5′-tagged DNA fragments for use in preparing templates fornext-generation or nucleic acid amplification, the kit comprising: atransposome composition comprising a transposase and a transposon endcomposition comprising (i) a transferred strand that has a 3′-portionthat exhibits the transferred transposon end sequence and a 5′-portionthat exhibits the sequence for a tag domain for use in a next-generationsequencing or amplification reaction, and (ii) a 5′-phosphate-containingnon-transferred strand that exhibits only the non-transferred transposonend sequence, wherein the transposase forms a complex with thetransposon end composition that is active in an in vitro transpositionreaction; and a reaction buffer that contains dimethylformamide in anamount that results in it being present in the in vitro transpositionreaction at a final concentration of 10%.

In some embodiments, the kit additionally comprises at least one otherenzyme component selected from among: a DNA polymerase that has 5′nuclease or strand-displacement activity; a DNA polymerase that lacks 5′nuclease activity, a template-dependent NAD ligase, and atemplate-independent ligase. In some embodiments, the at least one otherenzyme component is selected from among: FAILSAFE™ DNA polymerase mix;Taq DNA polymerase, Tfl DNA polymerase, T4 DNA polymerase, E. coli DNAligase, bacteriophage TS2126 thermostable RNA ligase, Mth Rn Ithermostable RNA ligase, and CIRCLIGASE™ thermostable ssDNA ligase.

In some preferred embodiments wherein the at least one enzyme in the kitis a template-dependent ligase (e.g., E. coli DNA ligase), a highproportion of the ligase molecules are adenylated and ATP is notprovided in the kit. In some embodiments wherein the at least one enzymein the kit is a template-dependent ligase (e.g., E. coli DNA ligase),the kit additionally comprises a ligation tagging oligonucleotidecomprising a 3′-portion and a 5′-portion, wherein the 3′-portionexhibits a sequence of a tag domain and the 5′-portion exhibits a randomsequence consisting of about three to about eight nucleotides. In somepreferred embodiments, the ligation tagging oligonucleotide comprises a5′-portion that exhibits a random sequence consisting of fournucleotides. In some other embodiments wherein the at least one enzymein the kit is a template-dependent ligase, the kit additionallycomprises a hairpin transposon end composition. In some embodimentswherein the hairpin transposon end composition has a 5′ end that isadenylated, less than 50% of the molecules composing thetemplate-dependent nucleic acid ligase provided in the kit areadenylated and no ATP or NAD is provided in the kit.

In some preferred embodiments wherein the at least one enzyme in the kitis a template-independent ligase, selected from among bacteriophageTS2126 thermostable RNA ligase, Mth Rn I thermostable RNA ligase, andCIRCLIGASE™ thermostable ssDNA ligase, the template-independent ligaseis provided in a highly adenylated form and ATP is not provided in thekit.

In one preferred embodiment of the kit, the transposome comprises awild-type or hyperactive Tn5 transposase or MuA transposase that isprovided at a concentration wherein the final concentration of thetransposome in the in vitro transposition reaction is at least 250 nM.In some other embodiments, the final concentrations of wild-type orhyperactive Tn5 transposome or MuA transposome is at least 500 nM.

One preferred embodiment is a kit for generating tagged circular ssDNAfragments using EZ-Tn5™ transposase and E. coli DNA ligase, the kitcomprising: (1) a wild-type or mutant form of Tn5 transposase (e.g.,EZ-Tn5™ transposase); (2) a transposon end composition that consists ofa transferred strand that exhibits the transferred transposon endsequence and a non-transferred strand that exhibits the non-transferredtransposon end sequence for EZ-Tn5 transposase; (3) EZ-Tn5 transposasereaction buffer; and (4) a template-independent nucleic acid ligase thatcan catalyze intramolecular ligation of ssDNA in the absence of aligation template (e.g., selected from among the RNA ligase fromthermophage TS2126 (U.S. Pat. No. 7,303,901); CIRCLIGASE™ thermostablessDNA ligase (EPICENTRE Biotechnologies, Madison, Wis., USA); and MthRNA ligase.1). In one preferred embodiment, the transposase in the kitis a wild-type or mutant form of Tn5 transposase (e.g., EZ-Tn5™transposase) at a concentration of greater than or equal to: about 5units per microliter; about 10-20 units per microliter; about 20-40units per microliter; about 40-60 units per microliter; about 60-80units per microliter; or about 80-100 units per microliter. In onepreferred embodiment of the kit comprising EZ-Tn5™ transposase and thetemplate-independent ligase, the EZ-Tn5 pMEDS transposon end compositioncomprises both an EZ-Tn5 pMETS transferred strand that has a5′-monophosphate group and an EZ-Tn5 pMENTS non-transferred strand thathas a 5′-monophosphate group.

In one preferred embodiment, the transposase in the kit is a wild-typeor mutant form of Tn5 transposase (e.g., EZ-Tn5™ transposase) at aconcentration of greater than or equal to: about 5 units per microliter;about 10-20 units per microliter; about 20-40 units per microliter;about 40-60 units per microliter; about 60-80 units per microliter; orabout 80-100 units per microliter. In one preferred embodiment of thekit comprising EZ-Tn5™ transposase and the template-independent nucleicacid ligase, the EZ-Tn5 pMEDS transposon end composition comprises bothan EZ-Tn5 METS transferred strand that has a 5′-monophosphate group andan EZ-Tn5 pMENTS non-transferred strand that has a 5′-monophosphategroup.

The methods, compositions and kits of the invention are useful forgenerating tagged circular DNA fragments or fantail dsDNA fragments ordi-tagged linear ssDNA fragments (and amplification products thereof)from target DNA from any source for genomic, subgenomic, or metagenomicanalysis (e.g., for use in making labeled target for microarrayanalysis; e.g., for analysis of copy number variation, for detection andanalysis of single nucleotide polymorphisms, and for finding genes fromenvironmental samples such as soil or water sources). The methods areuseful in a variety of processes, including processes for amplificationof the whole genome of one or more organisms, including one or moremicrobial or environmental organisms for which conditions for culture orgrowth are unknown (e.g., whole genome amplification or WGA), real-timePCR, emulsion PCR, comparative genomic hybridization (CGH), comparativegenomic sequencing (CGS), and for preparing DNA-specific probes (e.g.,chromosome-specific probes, e.g., chromosome paints) for applicationssuch as fluorescent in situ hybridization (FISH). In some embodiments,the methods are also used for generating templates for massivelyparallel DNA sequencing (so-called “next-generation sequencing”). Eachof these processes or applications finds uses for both research andmolecular diagnostic purposes.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The detailed description of exemplary embodiments of the invention ispresented in the following sections:

-   I. Fragmenting And Di-Tagging DNA Using A Transposase And A DNA    Polymerase-   III. Fragmentation, Tagging And Single-Primer Amplification Of    Target DNA-   II. Fragmenting and Tagging DNA Using A Transposase And A Ligase-   IV. Generation Of Tagged Circular Ss-DNA Fragments From Ds-Target    DNA-   Using A Transposase And A Ligase-   V. Fragmentation And Tagging Of Ds-DNA By In Vitro Transposition Of    Hairpin Transposon End Compositions    I. Fragmenting and Di-Tagging DNA Using a Transposase and a DNA    Polymerase

The present invention comprises methods, compositions and kits for usinga transposase to generate 5′-tagged fragments from target DNA comprisingor consisting of one or more double-stranded (dsDNA) molecules, and thenjoining a second tag that exhibits a different DNA sequence than thefirst tag to the 3′-ends of said 5′-tagged DNA fragments. The first tagexhibits the sequence of the transferred strand of the transposon endrecognized by the transposase, and optionally, also exhibits one or moreother sequences that are 5′-of the sequence of said transferredtransposon end. The second tag is joined to the 3′-ends of the 5′-taggedDNA fragments in vitro using a DNA polymerase without performing a PCRamplification reaction.

One method of the invention comprises: incubating a transposase and atransposon end with which it forms a transposition complex in an invitro transposition reaction with target DNA under conditions and forsufficient time wherein the transferred transposon end is joined to thetarget DNA, generating 5′-tagged DNA fragments that have a first tag ontheir 5′-ends; incubating the 5′-tagged DNA fragments with a DNApolymerase under DNA polymerization conditions, which conditions do notcomprise thermocycling, for sufficient time wherein a second tag thatexhibits a sequence that is different from the first tag is joined tothe 3′ ends of the 5′-tagged DNA fragments, generating 5′- and 3′-taggedDNA fragments. In some embodiments, the method further comprises thestep of amplifying the 5′- and 3′-tagged DNA fragments using a DNApolymerase and at least one primer that is complementary to the secondtag. The target DNA can comprise or consist of double-stranded DNA fromany in vivo or in vitro source, such as genomic DNA, subgenomic DNA,plasmid or other episomal-derived DNA or recombinant DNA therein, ordouble-stranded cDNA made by reverse transcription of RNA. Genomic DNAcan comprise or consist of one or more genomes from a biological orenvironmental source. Thus, the methods, compositions and kits of theinvention are useful for generating 5′- and 3′-tagged DNA fragments and,optionally, amplifying 5′- and 3′-tagged DNA fragments generated fromtarget DNA from any source for use in methods and applications known inthe art for genomic, subgenomic, or metagenomic analysis (e.g., foranalysis of copy number variation, single nucleotide polymorphisms, orother methods of genomic analysis. The methods are useful in a varietyof processes, including, but not limited to, metagenomic analysis ofgenomes of one or more organisms, including one or more microbial orenvironmental organisms for which conditions for culture or growth areunknown, real-time PCR, emulsion PCR, comparative genomic hybridization(CGH), comparative genomic sequencing (CGS). The methods areparticularly useful for generating templates for some types of massivelyparallel DNA sequencing (so-called “next-generation sequencing”).

Use of a Strand-Displacing DNA Polymerase and/or a DNA Polymerase with5′ Nuclease Activity for Generating DNA Fragments that have 5′ and 3′Tags that Exhibit the Sequences of Different Tranposon Ends

In some preferred embodiments, the present invention provides a methodfor generating a library of DNA fragments comprising 5′- and 3′-taggedDNA fragments from target DNA comprising or consisting of one or moredouble-stranded (dsDNA) molecules, the method comprising:

Providing:

-   -   1. target DNA comprising or consisting of one or more        double-stranded (dsDNA) molecules (e.g., eukaryotic and/or        prokaryotic genomic DNA or double-stranded cDNA prepared by        reverse transcription of RNA),    -   2. a transposase (e.g., a wild-type or mutant transposase; e.g.,        wild-type or mutant Tn5 transposase, e.g., EZ-Tn5™ transposase,        or e.g., HYPERMU™ MuA transposase, EPICENTRE Biotechnologies,        Madison, Wis., USA), and    -   3. a transposon end that is capable of forming a functional        complex with the transposase in a transposition reaction (e.g.,        the 19-bp outer end (“OE”) transposon end, inner end (“IE”)        transposon end, or “mosaic end” (“ME”) transposon end recognized        by a wild-type or mutant Tn5 transposase, e.g., by EZ-Tn5™        transposase, or the R1 and R2 transposon end, e.g., by HYPERMU™        MuA transposase), said transposon end comprising double-stranded        DNA consisting of a transferred strand and a non-transferred        strand, which, in combination, exhibit the sequences of the        double-stranded transposon end, wherein the transferred strand        exhibits the sequence of a first tag; and    -   4. a DNA polymerase that is strand-displaces or digests DNA that        is annealed to a template strand downstream of the 3′-end of the        DNA molecule that is being extended by said DNA polymerase        (i.e., the DNA polymerase has strand-displacement and/or 5′        nuclease activity; e.g., Taq DNA polymerase, Tfl DNA polymerase,        FAILSAFE™ DNA polymerase mix; phi29 DNA polymerase, E. coli DNA        polymerase I and DISPLACEACE™ DNA polymerase, all available from        EPICENTRE Biotechnologies, Madison, Wis., USA);

Incubating the target DNA with the transposase and the transposon endunder conditions and for sufficient time wherein transposase-catalyzedinsertion of the transposon end into both strands of the target DNAgenerates 5′-tagged DNA fragments, each of which has the first tag onits 5′-end (e.g., FIG. 2); and

Incubating 5′-tagged DNA fragments generated in the in vitrotransposition reaction with the DNA polymerase under conditions and forsufficient time wherein the DNA polymerase extends the 3′ ends of the5′-tagged DNA fragments, thereby joining a second tag that exhibits atleast a portion of the non-transferred transposon end sequence to the5′-tagged DNA fragments and generating 5′- and 3′-tagged DNA fragments.

In some preferred embodiments, the transferred strand exhibits only thetransferred transposon end sequence and, therefore, the first tag thatis present in the 5′-tagged DNA fragments exhibits only the transferredtransposon end sequence. In some other embodiments, the transferredstrand comprises or consists of a 3′-portion and a 5′-portion, whereinthe 3′-portion exhibits the sequence of the transferred transposon endand the 5′-portion exhibits any other desired sequence, in whichembodiments the first tag comprises or consists of both the 3′-portionand the 5′-portion. In embodiments wherein the transferred strandcomprises or consists of a 3′-portion and a 5′-portion, thenon-transferred strand may, but need not, exhibit a sequence that iscomplementary to the 5′-portion of the transferred strand.

In some embodiments wherein the transferred strand comprises or consistsof a 3′-portion and a 5′-portion, the 5′-portion exhibits a sequencingtag (e.g., the Roche 454A sequencing tag; as diagrammed, e.g., in FIG.10) and the 3′-portion exhibits the sequence of the transferred strandof the transposon end. This generates 5′-tagged DNA fragments with afirst tag that comprises or consists of a sequencing tag (e.g., theRoche 454A sequencing tag). Then, the DNA polymerase is used to join asecond tag that comprises or consists of the other sequencing tag (e.g.,the Roche 454B sequencing tag) to the 5′-tagged DNA fragments, therebygenerating a library of DNA fragments comprising 5′- and 3′-tagged DNAfragments with both sequencing tags (e.g., the 454A and 454B; as shownschematically in FIG. 10). The 5′- and 3′-tagged DNA fragments in thedesired size range are used as templates for next-generation sequencingusing the Roche 454 Genome Sequencer FLX System. In other embodiments,the 5′- and 3′-tagged DNA fragments in the library are generated withfirst and second tags that are appropriate as sequencing tags fornext-generation sequencing using any other sequencing platforms (e.g.,using the ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™sequencing platform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™sequencing platform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform,the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICSsequencing platform, the INTELLIGENT BIOSYSTEMS' sequencing platform, orthe HELICOS sequencing platform).

In some embodiments, multiple double-stranded transposon ends for oneparticular transposase or multiple different transposon ends recognizedby different transposase enzymes are used. In some preferred embodimentswherein a strand-displacing DNA polymerase or a DNA polymerase that has5′ nuclease activity is used, two different transposon ends are insertednear to each other in opposite strands of the target DNA, and then theDNA polymerase extends the 3′-ends of the 5′-tagged DNA fragments usingthe opposite strand as a template, thereby generating a library of 5′-and 3′-tagged DNA fragments with tags that exhibit different transposonend sequences on the 3′-end than on the 5′-end (e.g., wherein thetransferred strand of the first transposon end and the transferredstrand of the second transposon end are different and are joined toopposite strands of the target DNA (e.g., FIG. 7).

Thus, in some embodiments, the method additionally comprises:

Additionally providing:

-   -   5. a second transposase that recognizes a different transposon        end from the transposon end recognized by the first transposase        (referred to in this embodiment as the “first transposon end”        recognized by the “first transposase”); and    -   6. a second transposon end that is capable of forming a        functional complex with the second transposase in a        transposition reaction, said transposon end comprising a        transferred strand and a non-transferred strand, which, in        combination, exhibit the sequences of the double-stranded        transposon end, wherein the transferred strand exhibits a        sequence that is complementary to the sequence exhibited by the        second tag; and

Incubating the target DNA with the first transposase and the firsttransposon end and the second transposase and the second transposon endunder conditions and for sufficient time wherein the first and secondtransposase-catalyzed insertions of the first and second transposon endsinto the target DNA generates DNA fragments, each of which exhibits thesequence of the transferred strand of the first transposon end or thesecond transposon end on its 5′-end; and

Incubating the 5′-tagged DNA fragments with the DNA polymerase under DNApolymerization conditions, which conditions do not comprise denaturationof dsDNA or thermocycling, and for sufficient time wherein the DNApolymerase extends the 3′ end of the 5′-tagged DNA fragments, therebyjoining the second tag to the 5′-tagged DNA fragments and generating alibrary of tagged DNA fragments (e.g., 5′- and 3′-tagged DNA fragments)without performing an amplification reaction.

In some of embodiments, the method comprises simultaneously incubatingthe target DNA with both the first transposase and the first transposonend oligonucleotides and the second transposase and the secondtransposon end oligonucleotides in the same reaction mixture. In someother embodiments, the method is performed sequentially by firstincubating the target DNA with the first transposase and the firsttransposon end oligonucleotides and then incubating the products fromthat reaction with the second transposase and the second transposon endoligonucleotides. In some of the embodiments wherein the method isperformed sequentially, the products from the reaction of the target DNAwith the first transposase and the first transposon end oligonucleotidesare purified before incubating those products with the secondtransposase and the second transposon end oligonucleotides.

In some embodiments of the method wherein the transferred strandcomprises or consists of a 3′-portion and a 5′-portion, the 5′-portionexhibits the sequence of a sequencing tag (e.g., a first Roche 454sequencing tag, e.g., the Roche 454A) and the 3′-portion exhibits thesequence of the transferred strand of the transposon end. A “sequencingtag”, as used herein, means a tag that is joined to the 5′-end or 3′-endof a single-stranded DNA fragment generated from the target DNAmolecule, which tag is for the purpose of facilitating sequencing ofsaid DNA fragment. For example, in some embodiments, the sequencing tagprovides a site for capturing said DNA fragment strand on a surfaceand/or for priming DNA synthesis of said DNA fragment and/or thecomplement of said DNA fragment (e.g., as the Roche 454A and 454Bsequencing tags for the Roche 454 Genome Sequencer FLX System are used).Thus, when the 5′-portion of the transferred strand exhibits thesequence of a sequencing tag, the 5′-tagged DNA fragments have a firsttag that comprises or consists of the sequencing tag (e.g., the Roche454 sequencing tag). Then, the DNA polymerase joins a second tag thatcomprises or consists of a second sequencing tag (e.g., the Roche 454Bsequencing tag) to the 5′-tagged DNA fragments, thereby generating alibrary of 5′- and 3′-tagged DNA fragments with sequencing tags on eachend (e.g., the 454A and Roche 454B sequencing tags). The 5′- and3′-tagged DNA fragments in the library of the desired size range areused as templates for next-generation sequencing using the Roche 454Genome Sequencer FLX System. In other embodiments, 5′- and 3′-tagged DNAfragments are generated with first and second tags that are sequencingtags for next-generation sequencing using other sequencing platforms(e.g., using the ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™sequencing platform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™sequencing platform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform,the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICSsequencing platform, the INTELLIGENT BIOSYSTEMS' sequencing platform, orthe HELICOS sequencing platform).

In some embodiments, each of the different double-stranded transposonends comprises a different transferred strand that has a 5′ portion anda 3′ portion, wherein the 5′-portion of each different transferredstrand exhibits a different desired tag sequence and the 3′-portionexhibits the respective transferred transposon end sequence. In someembodiments, e.g., as shown in one example presented in FIG. 8, the 5′-and 3′-tagged DNA fragments in the library have both a first tag intheir 5′ end and a second tag in their 3′-end. The different transposonends shown in FIG. 8 have been inserted in different locations duringseparate in vitro transposition events catalyzed by the sametransposase. However, in some other embodiments, different transposasesthat form functional transposition complexes with different transposonends are used. The different tags in the 5′-portions of the transferredstrand of the each transposon end can exhibit any desired sequences forany desired purpose. By way of example, in some embodiments, the firsttag and second tag of the 5′- and 3′-tagged DNA fragments exhibit thesequences of the Roche 454A and 454B sequencing tags and, afterisolating the fragments in the desired size range, are used as templatesfor next-generation using the Roche 454 Genome Sequencer FLX System.Similarly, in other embodiments, the 5′- and 3′-tagged DNA fragments,after isolating those that are in the desired size range, are used astemplates for next-generation using another sequencing platform (e.g.,using the ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™sequencing platform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™sequencing platform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform,the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICSsequencing platform, the INTELLIGENT BIOSYSTEMS' sequencing platform, orthe HELICOS sequencing platform). In some preferred embodiments, the 5′-and 3′-tagged DNA fragments are generated using this method from targetDNA comprising a whole genome of a cell or organism.

In some embodiments, the transferred strand of the first transposon endor of the second transposon end is labeled with an affinity-bindingmolecule (e.g., biotin) or with a detectable molecule (e.g., afluorescent dye) that permits capture (e.g., using a surface to whichstreptavidin is bound for capture of the biotinylated molecules) ordetection of 5′- and 3′-tagged DNA fragments that have a tag with theaffinity-binding molecule or the detectable molecule at the 5′-end.

Adding a Tag Domain to 5′- and 3′-Tagged DNA Fragments: In someembodiments, a DNA polymerase and an oligonucleotide comprising atemplate for a tag domain is used to add a tag domain to the 3′-ends ofthe 5′- and 3′-tagged DNA fragments in the library of tagged DNAfragments (e.g., FIG. 19). In some embodiments, the DNA polymerase usedto add the tag domain is a thermostable DNA polymerase and theoligonucleotide is a PCR primer, and the tag domain is joined to thesecond tag by performing PCR.

III. Fragmentation, Tagging and Single-Primer Amplification of TargetDNA

One preferred method of the invention comprises: incubating atransposome complex consisting of a transposase and a transposon endwith which it forms a transposition complex with target DNA in an invitro transposition reaction under conditions and for sufficient timewherein the transferred transposon end inserts into multiple sites inboth strands of the target DNA; incubating the products of the in vitrotransposition reaction with a DNA polymerase that hasstrand-displacement and/or 5′-nuclease activity under conditions and forsufficient time wherein the 3′-end of each strand of target DNA that hasthe transferred transposon end joined to its 5′-end is extended usingthe opposite strand of the target DNA as a template, wherein each saidDNA polymerase-catalyzed extension displaces or digests thenon-transferred transposon end that is annealed to the next adjacenttransferred transposon end that is joined to the opposite strand of thetarget DNA, thereby generating a library of di-tagged DNA fragments,each comprising a different portion of the target DNA with a transferredstrand on its 5′-end and a non-transferred strand on its 3′-end, whereinthe population of all of the di-tagged DNA fragments is substantiallyrepresentative of the sequence of the target DNA from which they weregenerated; and incubating the library of di-tagged DNA fragments with athermostable DNA polymerase and a single primer that exhibits at least aportion of the transferred transposon end sequence under PCRthermocycling conditions, thereby generating an amplified library of thedi-tagged DNA fragments. In some preferred embodiments, the methodcomprises generating the amplified library of di-tagged DNA fragments inthe presence of one or more labeled dNTPs that are used as substrates bythe thermostable DNA polymerase, thereby generating an amplified libraryof labeled di-tagged DNA fragments.

Thus, one embodiment of the invention is an in vitro method forsingle-primer amplification of DNA fragments generated from a targetDNA, the method comprising: carrying out a transposition reaction in thepresence of a target DNA and in the presence of a transposon endconsisting of a transferred strand that exhibits the transferredtransposon end sequence and a non-transferred strand that exhibits thenon-transferred transposon end sequence, said transposition reactionresulting in multiple insertions comprising joining of the transferredtransposon end to each strand of the target DNA, thereby generating5′-tagged DNA fragments that are annealed to each other, each of whichhas a first tag on its 5′-end that exhibits the transferred transposonend sequence; extending the 3′-ends of the 5′-tagged DNA fragments withthe DNA polymerase that has strand-displacement and/or 5′ nucleaseactivity using the opposite strands to which the 5′-tagged DNA fragmentsare annealed as templates; and performing a PCR amplification reactionusing a single primer that exhibits at least a portion of thetransferred transposon end sequence, a thermostable DNA polymerase, andat least one labeled dNTP that is used as a substrate by thethermostable DNA polymerase, thereby generating the amplified library ofdi-tagged DNA fragments that are representative of the target DNA.

In some preferred embodiments, the target DNA is selected from amongeukaryotic and/or prokaryotic genomic DNA or double-stranded cDNAprepared by reverse transcription of RNA.

In some preferred embodiments, the transposome is a complex of awild-type or hyperactive mutant form of a transposase selected fromamong Tn5 transposase, MuA transposase, Sleeping Beauty transposase,Mariner transposase, Tn7 transposase, Tn10 transposase, Ty1 transposase,and Tn552 transposase and a transposon end with which the transposaseforms a complex that is active in a transposition reaction.

In some preferred embodiments, a single enzyme or enzyme mix is used asboth the DNA polymerase that has strand-displacement and 5′ nucleaseactivity and the thermostable DNA polymerase, which DNA polymerase ormix is selected from among wild-type or recombinant forms of TAQ DNApolymerase, Tfl DNA polymerase, Tth DNA polymerase, and FAILSAFE™ DNApolymerase mix.

In some preferred embodiments, the at least one labeled dNTP comprisinga label, e.g., a cyanine, (e.g., Cy5.5, Cy5, Cy3, Cy2), FITC, AlexaFluors (e.g., 647, 594), Texas Red, JOE, 5-FAM, 6-FAM, VIC, HEX, 6-ROX,Rhodamine, Lissamine, Cyan 500, etc. (See, e.g., Handbook of MolecularProbes, R. Haughland, Molecular Probe, Eugene, Oreg., incorporatedherein by reference).

In some preferred embodiments, the library or an amplified library 5′-and 3′-tagged DNA fragments generated using this method are from targetDNA comprising or consisting of a whole genome of a cell or organism. Insome embodiments, the library or an amplified library of di-tagged DNAfragments generated using this method are from target DNA comprising orconsisting of genomes and/or double-stranded cDNA from all organisms(e.g., multiple organisms) that are present in an environmental sample(e.g., for metagenomic or metatranscriptomic research or applications).

In some preferred embodiments of the method, the transferred strandexhibits only the transferred transposon end sequence and, therefore,the first tag that is present in the 5′-tagged DNA fragments exhibitsonly the transferred transposon end sequence. In some other embodiments,the transferred strand comprises or consists of a 3′-portion and a5′-portion, wherein the 3′-portion exhibits the transferred transposonend sequence and the 5′-portion exhibits any other desired nucleotide ornucleotide sequence, in which embodiments the first tag comprises orconsists of both the 3′-portion and the 5′-portion. In embodimentswherein the transferred strand comprises or consists of a 3′-portion anda 5′-portion, the non-transferred strand may, but need not, exhibit asequence that is complementary to the 5′-portion of the transferredstrand. In some preferred embodiments wherein the transferred strandcomprises or consists of a 3′-portion and a 5′-portion, the 5′-portionexhibits at least one nucleotide that comprises a capture domain (e.g.,a nucleotide that comprises a biotin moiety, which can be captured by astreptavidin moiety which is bound to a surface; or e.g., anotheraffinity-binding molecule).

II. Fragmenting and Tagging DNA Using a Transposase and a Ligase

The present invention comprises methods, compositions and kits for usinga transposase to generate 5′-tagged fragments from target DNA comprisingor consisting of one or more double-stranded (dsDNA) molecules, and thenjoining a second tag that exhibits a different DNA sequence than thefirst tag to the 3′-ends of said 5′-tagged DNA fragments. The first tagexhibits the sequence of the transferred strand of the transposon endrecognized by the transposase, and optionally, also exhibits one or moreother sequences that are 5′-of the sequence of said transferredtransposon end. The second tag is joined to the 3′-ends of the 5′-taggedDNA fragments in vitro using a nucleic acid ligase.

One method of the invention comprises: incubating a transposase and atransposon end with which it forms a transposition complex in an invitro transposition reaction with target DNA under conditions and forsufficient time wherein the transferred transposon end is joined to thetarget DNA, generating 5′-tagged DNA fragments that have a first tag ontheir 5′-ends; incubating the 5′-tagged DNA fragments with a nucleicacid ligase and a ligation tagging oligonucleotide that comprises orconsists of a second tag under conditions and for sufficient timewherein the ligation tagging oligonucleotide is joined to the 3′ ends ofthe 5′-tagged DNA fragments, generating a library of 5′- and 3′-taggedDNA fragments. In some embodiments, the method further comprises thestep of amplifying the library of 5′- and 3′-tagged DNA fragments usinga DNA polymerase and at least one primer that is complementary to thesecond tag. In some embodiments, the step of amplifying the library of5′- and 3′-tagged DNA fragments using a DNA polymerase comprises PCRamplification using a thermostable DNA polymerase, a first PCR primerthat is complementary to the second tag, and a second PCR primer thatexhibits a sequence that is identical to at least a portion of thesequence exhibited by the first tag.

One preferred embodiment of the present invention is a method forgenerating a library of tagged DNA fragments comprising 5′- and3′-tagged DNA fragments from target DNA comprising or consisting of oneor more double-stranded (dsDNA) molecules, the method comprising:

Providing:

-   -   1. target DNA comprising or consisting of one or more        double-stranded (dsDNA) molecules (e.g., eukaryotic and/or        prokaryotic genomic DNA or double-stranded cDNA prepared by        reverse transcription of RNA),    -   2. a transposase (e.g., a wild-type or mutant transposase; e.g.,        wild-type or mutant Tn5 transposase, e.g., EZ-Tn5™ transposase,        or, e.g., HYPERMU™ MuA transposase, EPICENTRE Biotechnologies,        Madison, Wis., USA), and    -   3. a transposon end that is capable of forming a functional        complex with the transposase in a transposition reaction (e.g.,        the 19-bp outer end (“OE”) transposon end, inner end (“IE”)        transposon end, or “mosaic end” (“ME”) transposon end recognized        by a wild-type or mutant Tn5 transposase, e.g., by EZ-Tn5™        transposase, or the R1 and R2 transposon end, e.g., by HYPERMU™        MuA transposase), said transposon end comprising double-stranded        DNA consisting of a transferred strand and a non-transferred        strand, which, in combination, exhibit the sequences of the        double-stranded transposon end, wherein the transferred strand        exhibits the sequence of a first tag,    -   4. a ligation tagging oligonucleotide that has a 5′-end that is        capable of being ligated to the 3′ hydroxyl of a DNA molecule        and that exhibits a sequence of a second tag, and    -   5. a nucleic acid ligase;

Incubating the target DNA with the transposase and the transposon endunder conditions and for sufficient time wherein transposase-catalyzedinsertion of the transposon end into the target DNA generates 5′-taggedDNA fragments, each of which has the first tag on its 5′-end; and

Incubating the 5′-tagged DNA fragments with the nucleic acid ligase andthe ligation tagging oligonucleotide under conditions and for sufficienttime wherein the second tag is joined to their 3′-ends and a library of5′- and 3′-tagged DNA fragments is generated, each of which tagged DNAfragments has the first tag on the 5′ end and the second tag on the 3′end.

In some preferred embodiments, the transferred strand exhibits only thetransferred transposon end sequence and, therefore, the first tag thatis present in the tagged DNA fragments exhibits only the transferredtransposon end sequence. In some other embodiments, the transferredstrand comprises or consists of a 3′-portion and a 5′-portion, whereinthe 3′-portion exhibits the transferred transposon end sequence and the5′-portion exhibits any other desired sequence, in which embodiments thefirst tag comprises or consists of both the 3′-portion and the5′-portion. In embodiments wherein the transferred strand comprises orconsists of a 3′-portion and a 5′-portion, the non-transferred strandmay, but need not, exhibit a sequence that is complementary to the5′-portion of the transferred strand.

In some embodiments wherein the transferred strand comprises or consistsof a 3′-portion and a 5′-portion, the 5′-portion exhibits the sequenceof a sequencing tag (e.g., a first Roche 454 sequencing tag, e.g., theRoche 454A) and the 3′-portion exhibits the sequence of the transferredstrand of the transposon end. A “sequencing tag”, as used herein, meansa tag that is joined to the 5′-end or 3′-end of a single-stranded DNAfragment generated from the target DNA molecule, which tag is for thepurpose of facilitating sequencing of said DNA fragment. For example, insome embodiments, the sequencing tag provides a site for capturing saidDNA fragment strand on a surface and/or for priming DNA synthesis ofsaid DNA fragment or the complement of said DNA fragment (e.g., as theRoche 454A and 454B sequencing tags for the Roche 454 Genome SequencerFLX System are used). Thus, when the 5′-portion of the transferredstrand exhibits the sequence of a sequencing tag, the 5′-tagged DNAfragments have a first tag that comprises or consists of the sequencingtag (e.g., the Roche 454 sequencing tag). Then, the nucleic acid ligaseligates a ligation tagging oligonucleotide that has a second tagcomprising or consisting of a second sequencing tag (e.g., the Roche454B sequencing tag) to the 5′-tagged DNA fragments, thereby generatingthe library of 5′- and 3′-tagged DNA fragments with sequencing tags oneach end (e.g., the 454A and Roche 454B sequencing tags). The 5′- and3′-tagged DNA fragments are generated to have a desired size rangeappropriate for use as templates for next-generation sequencing usingthe Roche 454 Genome Sequencer FLX System. In other embodiments, alibrary of tagged DNA fragments is generated comprising tagged DNAfragments of a size and with first and second tags that are appropriatefor use as sequencing tags for next-generation sequencing using anothersequencing platform (e.g., using the ROCHE 454 sequencing platform, theILLUMINA™ SOLEXA™ sequencing platform, the LIFE TECHNOLOGIES/APPLIEDBIOSYSTEMS' SOLID™ sequencing platform, the PACIFIC BIOSCIENCES' SMRT™sequencing platform, the POLLONATOR Polony sequencing platform, theCOMPLETE GENOMICS sequencing platform, the INTELLIGENT BIOSYSTEMS'sequencing platform, or the HELICOS sequencing platform).

Template-Dependent Ligation of the Second Tag

In some preferred embodiments, the method comprises providing a ligationtagging oligonucleotide that comprises or consists of a 3′-portion and a5′-portion, wherein the 3′-portion exhibits a second tag that comprisesor consists of any sequence that is desired to be joined to the 3′-endof the 5′-tagged DNA fragments (i.e., an arbitrary sequence) and the5′-portion has a 5′-monophosphate group and exhibits a random sequence(e.g., a random sequence consisting of about three to about eightnucleotides) at its 5′-end. In some preferred embodiments, the ligationtagging oligonucleotide has a 5′-portion that exhibits a random sequenceof four nucleotides (e.g., in some embodiments described herein whereinthe transposase is a wild-type or mutant Tn5 transposase (e.g., EZ-Tn5™transposase, or e.g., HYPERMU™ MuA transposase, EPICENTREBiotechnologies, Madison, Wis., USA) and the nucleic acid ligase is E.coli DNA ligase).

The invention is not limited to a ligation tagging oligonucleotide thathas a 5′-portion that exhibits a random sequence consisting of aboutthree to about eight nucleotides. For example, the invention alsoincludes methods wherein the ligation tagging oligonucleotide has a5′-portion that: exhibits a random sequence consisting of only twonucleotides; exhibits a random sequence consisting of greater than eightnucleotides; exhibits a semi-random sequence rather than a totallyrandom sequence; or that exhibits a sequence comprising one or moredegenerate nucleotides (e.g., an inosine nucleotide) rather than atotally random sequence. However, a ligation tagging oligonucleotidethat has a 5′-portion that exhibits a random sequence consisting ofabout three to about eight nucleotides is preferred.

In some preferred embodiments, ligation of the ligation taggingoligonucleotide to the 3′ end of the 5′-tagged DNA fragments occurs onlyin the presence of a DNA template that exhibits a sequence that isexactly complementary to the ligation junction; in such embodiments, thetemplate to which the two nucleic acid molecules that are ligated annealis referred to herein as a “ligation template” and the ligation isreferred to as “template-dependent ligation”. In some embodimentswherein the ligation occurs only in the presence of a ligation template,the nucleic acid ligase is a DNA ligase that requires a ligationtemplate, and is referred to herein as a “template-dependent ligase”(e.g., an NAD-type template-dependent DNA ligase such as, but notlimited to, E. coli DNA ligase, Tth DNA ligase, Tfl DNA ligase, orAMPLIGASE® DNA ligase, which are available from EPICENTREBiotechnologies, Madison, Wis., USA). In some other embodiments whereinthe ligation occurs only in the presence of a ligation template, thenucleic acid ligase is a DNA ligase which, while it does not require aligation template for ligation, it catalyzes ligation more efficientlyin the presence of the ligation template than in its absence (e.g., anATP-type template-dependent DNA ligase such as, but not limited to, T4DNA ligase or FASTLINK™ DNA ligase, which are available from EPICENTREBiotechnologies, Madison, Wis., USA). If the ligation occurs on aligation template, the ligation is referred to as “template-dependentligation” herein, even if the ligase could also catalyzetemplate-independent ligation. In preferred embodiments, the randomsequence of the ligation tagging oligonucleotide is short, in whichembodiments a nucleic acid that catalyzes template-dependent ligase at alower temperature (e.g., less than or equal to about 40° C., less thanor equal to about 37° C., less than or equal to about 30° C., less thanor equal to about 25° C., or less than or equal to about 20° C.) ispreferred. In some preferred embodiments, the template-dependent ligaseis E. coli DNA ligase.

The invention is not limited with respect to the ligation method usedexcept that, with respect to embodiments comprising template-dependentligation, the ligation should occur efficiently in the presence of atarget sequence to which the ligation tagging oligonucleotide and the5′-tagged DNA fragments anneal contiguously and ligation should occurrarely or not at all in the absence of a target sequence. As usedherein, “template-dependent ligation” refers to any suitable method forjoining adjacent 5′- and 3′-ends of ligation tagging oligonucleotidesand 5′-tagged DNA fragments, respectively, that are adjacent to orcontiguous to or that abut each other when annealed to a targetsequence.

Transposase-catalyzed insertion of the transposon end into both strandsof the target DNA results in fragmentation of the target DNA, joining ofthe transferred transposon end to each strand of the target DNA, andgeneration of a 9-base region of single-stranded target DNA 3′-of thesite of joining of the transferred transposon end, which results in agap-region in the opposite strand of the target DNA (e.g., FIG. 3). Thesingle-stranded region of target DNA downstream of the transferredtransposon end can serve as a ligation template for annealing of therandom portion of the ligation tagging oligonucleotide. Among all of thesequences represented by the ligation tagging oligonucleotide thatexhibits the random sequence in its 5′ portion (which includes allpossible sequences), at least one of them exhibits a sequence at its5′-end that is capable of annealing to each single-stranded region inthe target DNA so that the 5′-phosphorylated end of that ligationtagging oligonucleotide is adjacent to and abuts the 3′-end of thetarget DNA that is complementary to the 5′-tagged DNA fragment, in whichevent, the nucleic acid ligase can then catalyze template-dependentligation of the ligation tagging oligonucleotide to said 3′-end. In someembodiments, the transferred strand of the transposon end is insertedinto opposite strands of the target DNA at two locations that are inrelatively close proximity, generating two 5′-tagged DNA fragments asshown in FIG. 2, and two 5′- and 3′-tagged DNA fragments as shown inFIG. 7. Upon denaturation of the two 5′- and 3′-tagged DNA fragments,they can be used for a variety of applications, including foramplification (e.g., by PCR using a first PCR primer that iscomplementary to the second tag and a second PCR primer that iscomplementary to the first tag).

In some embodiments of the method comprising using a nucleic acid ligaseand template-dependent ligation to join a second tag to the 5′-taggedDNA fragments, the 5′- and 3′-tagged DNA fragments comprise a 5′ firsttag that exhibits a transposon end sequence and a 3′ second tag thatdoes not exhibit a transposon end sequence, (although the 3′-portion ofligation tagging oligonucleotide can comprise or consist of a second tagthat exhibits any desired sequence, including, if desired, a transposonend sequence). For example, in some embodiments, the 3′-portion of theligation tagging oligonucleotide exhibits the sequence of the Roche 454sequencing tag or of a sequencing tag for another sequencing platform(e.g., using the ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™sequencing platform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™sequencing platform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform,the POLLONATOR Polony sequencing platform, the COMPLETE GENOMICSsequencing platform, the INTELLIGENT BIOSYSTEMS' sequencing platform, orthe HELICOS sequencing platform).

By way of further example, in some embodiments, the second tag in the3′-portion of the ligation tagging oligonucleotide exhibits the sequenceof an RNA polymerase promoter (e.g., a T7-type RNA polymerase promoter;e.g., a T7, T3, SP6, or phage N4 MINI-V™ RNA polymerase promoter;EPICENTRE Biotechnologies, Madison, Wis., USA); in general, if the RNApolymerase requires a double-stranded RNA polymerase promoter, thesecond tag in these embodiments exhibits the “sense RNA polymerasepromoter sequence”, meaning the sequence of the RNA polymerase promoterthat is joined to the 3′-end of the template DNA strand that istranscribed by the RNA polymerase, in which embodiments, thecomplementary “anti-sense RNA polymerase promoter sequence” must also beprovided or synthesized during a step in the method to generate adouble-stranded RNA polymerase promoter that will be recognized by theRNA polymerase. In some embodiments, the second tag in the 3′-portion ofthe ligation tagging oligonucleotide also exhibits the sequence of an“address tag”, meaning a sequence that permits identification of aspecific sample (e.g., by using an address tag in a ligation taggingoligonucleotide that exhibits a different sequence for each target DNAsample). In some embodiments, the 3′-portion of the ligation taggingoligonucleotide also exhibits the sequence of one or more other tags fora particular purpose in the method.

In some preferred embodiments, the 5′ portion of the ligation taggingoligonucleotide is a random sequence of a length that is capable ofannealing to the single-stranded portions of the 5′-tagged DNA fragmentsgenerated from transposase-catalyzed insertion of the transposon endsinto both strands of the target DNA. The random sequence in the5′-portion of the ligation tagging oligonucleotide anneals to thissingle-stranded gap region adjacent to the 3′-end of the 5′-tagged DNAfragments, which serves as a ligation template for template-dependentligation of the ligation tagging oligonucleotide to the 3′-ends of thecomplementary strand of target DNA. In embodiments using EZ-Tn5™transposase, insertion of the 19-bp EZ-Tn5™ transposon endoligonucleotides into both strands of the target DNA generates 5′-taggedDNA fragments that exhibit 9-base gaps consisting of single-strandedregions that are opposite the sites of insertion the transferredtransposon end. However, the size of the gap wherein the ligationtagging oligonucleotide can anneal varies for different transposaseenzymes. For example, the MuA transposase generates a single-strandedregion of target DNA downstream of the site of insertion of thetransferred transposon end that is only five nucleotides. In someembodiments, the random sequence of the ligation tagging oligonucleotidecomprises or consists of between about three and about eight randomnucleotides. However, the length of the random sequence portion of theligation tagging oligonucleotide can vary for different transposaseenzymes based on the size of the single-stranded region generated andother factors, such as the length of the random sequence that is mostefficiently ligated with the respective nucleic acid ligase and ligationconditions used. For example, the Applicants observed that, using5′-tagged DNA fragments generated using EZ-Tn5™ transposase, a ligationtagging oligonucleotide with a 5′-portion consisting of a randomsequence of four nucleotides generated good yields of 5′- and 3′-taggedDNA fragments using E. coli DNA ligase as the nucleic acid ligase.However, this ligation tagging oligonucleotide with a 5′-portionconsisting of a random sequence of four nucleotides was not efficientlyligated by thermostable DNA-dependent ligases, such as AMPLIGASE®thermostable ligase under similar ligation conditions. In preferredembodiments, the 5′- and 3′-tagged DNA fragments comprise or consist ofall DNA fragments generated from the DNA sample.

In some embodiments, the nucleic acid ligase for template-dependentligation is a ligase that uses NAD as a co-factor. In some embodiments,the nucleic acid ligase for template-dependent ligation is selected fromamong the following NAD-type DNA ligases: E. coli DNA ligase, Tth DNAligase, Tfl DNA ligase, and Ampligase® DNA ligase (all available fromEPICENTRE Biotechnologies, Madison, Wis., USA), and Tsc DNA ligase(Roche Applied Systems, Indianapolis, Ind., USA). In some embodiments,the nucleic acid ligase for template-dependent ligation is an ATP-typeDNA ligase. In some embodiments, the ATP-type DNA ligase is selectedfrom among: T4 DNA ligase and FASTLINK™ DNA ligase (EPICENTREBiotechnologies, Madison, Wis., USA). In some preferred embodiments, thenucleic acid ligase is selected from among E. coli DNA ligase or anothermesophilic bacterial DNA ligase that uses NAD as a co-factor. In somepreferred embodiments, size-selection and purification of thesize-selected 5′-tagged DNA fragments is performed to improved theefficiency of ligation of the ligation tagging oligonucleotide to the5′-tagged DNA fragments using the DNA template-dependent DNA ligase(e.g., E. coli DNA ligase).

Template-Independent Ligation of the Second Tag

In some embodiments of the method comprising joining the second tag tothe 3′ end of the 5′-tagged DNA fragments using a nucleic acid ligase,the ligation tagging oligonucleotide that exhibits the second tag isligated directly to the 3′-end of the 5′-tagged DNA fragments withoutannealing the ligation tagging oligonucleotide to a ligation templateadjacent to the 3′-ends of the 5′-tagged DNA fragments. In theseembodiments, the ligation tagging oligonucleotide does not exhibit arandom sequence, but rather exhibits only the sequence of the second tagthat it is desired to be joined to the 5′-tagged DNA fragments. In theseembodiments, the ligation tagging oligonucleotide is ligated directly tothe 3′-ends of single-stranded 5′-tagged DNA fragments without using aligation template. In these embodiments of the method, the nucleic acidligase is a nucleic acid ligase that is capable of ligating asingle-stranded DNA molecule that has a 3′-hydroxyl group to asingle-stranded DNA molecule that has a 5′-monophosphate group in theabsence of annealing to a complementary sequence at the ligationjunction (e.g., selected from among T4 RNA ligase 1, T4 RNA ligase 2,bacteriophage TS2126 thermostable RNA ligase, and CIRCLIGASE™ DNAligase, EPICENTRE Biotechnologies, Madison, Wis., USA); and the methodadditionally comprises the step of: denaturing dsDNA comprising the5′-tagged DNA fragments prior to incubating the 5′-tagged DNA fragmentswith the nucleic acid ligase and the ligation tagging oligonucleotide.

The invention is not limited to a particular nucleic acid ligase and themethods comprising incubating the 5′-tagged DNA fragments with a nucleicacid ligase under conditions and for sufficient time wherein the secondtag is joined to their 3′-ends and a library of 5′- and 3′-tagged DNAfragments is generated will be understood to also comprise use of othercompositions in place of the nucleic acid ligase for template-dependentor a template-independent joining. By way of example, other ligationmethods such as, but not limited to, use of a ligation taggingoligonucleotide that comprises a topoisomerase moiety, wherein theligation comprises topoisomerase-mediated ligation (e.g., U.S. Pat. No.5,766,891, incorporated herein by reference) can be used, althoughtopoisomerase-mediated ligation is not preferred in most embodiments.

IV. Generation of Tagged Circular Ss-DNA Fragments from Ds-Target DNAUsing a Transposase and a Ligase (30842)

The present invention comprises methods, compositions and kits forgenerating a library comprising a population of tagged circular ssDNAfragments from target DNA in a sample for use as templates in DNAsequencing or nucleic acid amplification reactions. In general, eachtagged circular ssDNA fragment in the library exhibits a contiguoussequence of a portion of the target DNA and of a tag.

Briefly, in certain embodiments, the method comprises: incubating thetarget DNA, which is generally dsDNA, with a transposase and atransposon end composition in an in vitro transposition reaction tosimultaneously fragment and tag the target DNA, thereby generating apopulation of tagged DNA fragments; then denaturing the tagged DNAfragments to generate 5′-tagged ssDNA fragments, and then incubating the5′-tagged ssDNA fragments with a template-independent or non-homologousnucleic acid ligase that is capable of catalyzing template-independentintramolecular ligation (i.e., circularization) of ssDNA to generate alibrary of tagged circular ssDNA fragments. In some embodiments, thetagged circular ssDNA fragments are linearized by annealing anoligodeoxyribonucleotide that anneals to a restriction site within thetag, and then treating with the restriction endonuclease to generatelinear ssDNA fragments that have a portion of the tag on their 5′-endsand the remaining portion of the tag on their 3′-ends (which linearssDNA fragments are referred to herein as “di-tagged linear ssDNAfragments” or simply, “di-tagged ssDNA fragments”).

In some embodiments the tagged circular ssDNA fragments or the di-taggedlinear ssDNA fragments are used as DNA templates in nucleic acidamplification and/or DNA sequencing reactions. In some embodiments, themethod further comprises the step of amplifying and/or sequencing thelibrary of the tagged circular ssDNA fragments (e.g., to amplify ordetermine the sequence of the target DNA). In some embodiments, themethod further comprises the step of sequencing DNA that iscomplementary to the target DNA obtained by amplification of the taggedcircular ssDNA fragments or the di-tagged linear ssDNA fragments. Insome embodiments, at least a portion of the target DNAs in each of thetagged circular ssDNA fragments or the di-tagged linear ssDNA fragmentsis sequenced using a DNA polymerase and at least one primer that iscomplementary to the tag (e.g., for sequencing by synthesis). In someembodiments, at least a portion of the target DNAs in each of the taggedcircular ssDNA fragments or the di-tagged linear ssDNA fragments issequenced using a template-dependent ligase to ligate at least oneoligodeoxyribonucleotide that is complementary to the tag and at leastone other oligodeoxyribonucleotide that anneals to the portion of thetarget sequence (e.g., sequencing by ligation). In some embodiments, atleast a portion of the target DNA in each of the tagged circular ssDNAfragments or the di-tagged linear ssDNA fragments is sequenced byannealing oligodeoxyribonucleotides that anneal or hybridize to the tagand to a portion of the target sequence (e.g., sequencing byhybridization). In some embodiments, DNA that is complementary to thetagged circular ssDNA fragments or the di-tagged linear ssDNA fragmentsis sequenced using sequencing by synthesis, sequencing by ligation, orsequencing by hybridization.

Thus, one preferred embodiment of the present invention is a method forgenerating a library comprising a population of tagged circular ssDNAfragments from target DNA in a sample for use as templates in DNAsequencing or nucleic acid amplification reactions, each of which taggedcircular ssDNA fragments exhibits the sequence of a portion of thetarget DNA and the sequence of a tag that is joined to the portion ofthe target sequence, the method comprising:

Providing:

-   -   1. target DNA comprising or consisting of one or more        double-stranded (dsDNA) molecules (e.g., eukaryotic and/or        prokaryotic genomic DNA or double-stranded cDNA prepared by        reverse transcription of RNA using an RNA-dependent DNA        polymerase or reverse transcriptase to generate first-strand        cDNA and then extending a primer annealed to the first-strand        cDNA to generate dsDNA),    -   2. a transposase (e.g., a wild-type or mutant transposase; e.g.,        wild-type or mutant Tn5 transposase, e.g., EZ-Tn5™ transposase,        e.g., HYPERMU™ MuA transposase, EPICENTRE Biotechnologies,        Madison, Wis., USA), and    -   3. a transposon end composition that is capable of forming a        functional complex with the transposase in a transposition        reaction (e.g., comprising or consisting of the 19-bp outer end        (“OE”) transposon end, the 19-bp inner end (“IE”) transposon        end, or the 19-bp “mosaic end” (“ME”) transposon end recognized        by a wild-type or mutant Tn5 transposase, e.g., by EZ-Tn5™        transposase), said transposon end composition comprising or        consisting of a transferred strand and a non-transferred strand,        which, in combination, exhibit the sequences of the        double-stranded transposon end, wherein the transferred strand        exhibits the sequence of the tag,    -   4. a template-independent or non-homologous nucleic acid ligase        that is capable of template-independent intramolecular ligation        or circularization of ssDNA that has a 5′-monophosphate and a        3′-hydroxyl group (e.g., phage TS2126 thermostable RNA ligase,        e.g., wherein a high proportion of the RNA ligase molecules are        adenylated);

Incubating the target DNA with the transposase and the transposon endcomposition under conditions and for sufficient time whereintransposase-catalyzed insertion of the transferred strand into thetarget DNA generates 5′-tagged DNA fragments (e.g., FIG. 2); and

Denaturing the target DNA comprising 5′-tagged DNA fragments to obtain5′-tagged ssDNA fragments; and

Incubating the 5′-tagged ssDNA fragments with the nucleic acid ligaseunder conditions and for sufficient time wherein the 5′-tagged ssDNAfragments are intramolecularly ligated to generate a library of taggedcircular ssDNA fragments, each of which exhibits the sequence of aportion of the target DNA and of the tag.

In some embodiments, prior to the ligation step, the method additionallycomprises one or more steps to remove target DNA that is not taggedduring the transposition reaction and/or to remove components of thetransposon end composition that are not joined to target DNA.

In some embodiments, the method additionally comprises treating thelibrary containing the tagged circular ssDNA fragments with exonucleaseI to remove unligated linear ssDNA. In some embodiments, the methodadditionally comprises the step of treating the reaction mixture withexonuclease I and exonuclease III (EPICENTRE Biotechnologies, Madison,Wis.) to remove unligated linear ssDNA. Exonuclease III aids in removingsome linear ssDNA by digesting double-stranded regions of linear ssDNAmolecules that result from intramolecular or intermolecular annealing.In some preferred embodiments, the method additionally comprisestreating the library of tagged circular ssDNA fragments with T5exonuclease (EPICENTRE Biotechnologies, Madison, Wis.) to removeunligated linear ssDNA and dsDNA (e.g., DNA fragments that are nickedand/or contain single-stranded regions).

In some embodiments, the method further comprises amplifying the taggedcircular ssDNA fragments or the di-tagged linear ssDNA fragments bytranscription, the method comprising: (a) annealing to the sensepromoter sequence an oligodeoxyribonucleotide that exhibits acomplementary anti-sense promoter sequence, or, annealing to the taggedcircular ssDNA fragments or the di-tagged linear ssDNA fragments, aprimer that is complementary thereto and extending the primer with a DNApolymerase under conditions wherein a double-stranded RNA polymerasepromoter is synthesized; and (b) incubating the dsDNA products with anRNA polymerase that binds the RNA polymerase promoter under conditionswherein RNA is synthesized.

In some preferred embodiments wherein the transferred strand or a PCRprimer exhibits an RNA polymerase promoter sequence, the RNA polymerasepromoter is a T7-type RNA polymerase promoter and the method furthercomprises the step of transcribing the tagged circular ssDNA fragmentsin vitro using a T7-type RNA polymerase that recognizes the promoter.Most preferably, the RNA polymerase and promoter are chosen from amongT7 RNAP, T3 RNAP and SP6 RNAP and the corresponding cognate promoters.However, transcription steps of a method of the invention can use anyRNAP for which a suitable promoter sequence that permits transcriptionwith high specificity is known or can be obtained. Kits and enzymes forin vitro transcription are commercially available from many vendors andthe appropriate reaction mixtures and conditions for carrying out stepsof the present invention comprising in vitro transcription can use thoseproducts as described by the manufacturers. For example, in vitrotranscription using T7 RNAP can be carried out using the AMPLISCRIBE™T7-FLASH™ Transcription Kit or the AMPLISCRIBE™ T7 High YieldTranscription Kit from EPICENTRE Biotechnologies, Madison, Wis. asdescribed in the product literature. Similarly, if T3 RNAP or SP6 RNAPis used in a method of the invention for in vitro transcription, anAMPLISCRIBE™ T3-FLASH™ High Yield Transcription Kit or with theAMPLISCRIBE™ SP6 High Yield Transcription Kit (EPICENTREBiotechnologies, Madison, Wis.), respectively, can be used as described.

In some embodiments, the transferred strand, the ligation taggingoligonucleotide, or a PCR primer exhibits, in addition to the RNApolymerase promoter sequence, additional sequences for translation, suchas but not limited to a ribosome binding site and a translation startcodon (also referred to as a “translation start signal”), and the methodadditionally comprises translating the transcribed RNA. In some of theseembodiments, the method further comprises the step in vitro translationof the resulting RNA transcripts. Systems and kits for in vitrotranslation of the RNA transcripts are also commercially available frommany sources and can be used for the present invention. For example,rabbit reticulocyte lysate, wheat germ extract, and E. coli S30 extractsystems from Promega Corporation, Madison, Wis. can be used for thepresent invention. Still further, kits for coupled in vitrotranscription and in vitro translation are also commercially availableand can be used, such as TNT® Quick Coupled Transcription/TranslationSystems from Promega.

In some other embodiments, the method further comprises the step ofamplifying and/or sequencing the target DNA in the tagged circular ssDNAfragments using a DNA polymerase and at least one primer that iscomplementary to the tag. In some embodiments, the step of amplifyingthe tagged circular ssDNA fragments using a DNA polymerase comprisesrolling circle replication. In some embodiments, the step of amplifyingthe tagged circular ssDNA fragments using a DNA polymerase comprises PCRamplification using a thermostable DNA polymerase and a first PCR primerthat is complementary to at least a portion of the tag and a second PCRprimer that is complementary to at least a portion of the complement ofthe tag. In some embodiments, the method further comprises the step ofamplifying the tagged circular ssDNA fragments using an RNA polymerase.

Thus, in some other embodiments, the method further comprises amplifyingthe tagged circular ssDNA fragments by rolling circle replication (RCR),the method comprising: (a) annealing a primer that is complementary tothe tagged circular ssDNA fragments; and (b) extending the primerannealed to the tagged circular ssDNA fragments using astrand-displacing DNA polymerase (e.g., phi29 DNA polymerase or rBst DNApolymerase large fragment (EPICENTRE) or DISPLACEACE™ DNA polymerase(EPICENTRE). In these embodiments, the RCR amplification products areconcatameric ssDNA molecules that are complementary to the taggedcircular ssDNA fragments. In some embodiments wherein the taggedcircular ssDNA fragments exhibit an anti-sense promoter sequence, theconcatameric RCR amplification products exhibit a sense promotersequence and the method further comprises making the RNA polymerasepromoter double-stranded (e.g., by annealing to the sense promotersequence a complementary oligodeoxyribonucleotide that exhibits ananti-sense promoter sequence, and then transcribing the concatameric DNAusing an RNA polymerase that binds to the double-stranded RNA polymerasepromoter and initiates transcription therefrom.

In some preferred embodiments, the transposon end composition comprisesa transferred strand that exhibits only the transferred transposon endsequence and, therefore, the tag exhibits only the transferredtransposon end sequence. In some other embodiments, the transposon endcomposition comprises a transferred strand that comprises or consists ofa 3′-portion and a 5′-portion, wherein the 3′-portion exhibits thetransferred transposon end sequence and the 5′-portion exhibits anyother desired sequence, in which embodiments the tag comprises orconsists of both the 3′-portion and the 5′-portion. In some embodimentswherein the transposon end composition comprises a transferred strandthat comprises or consists of a 3′-portion and a 5′-portion, thenon-transferred strand exhibits a sequence that is complementary to the5′-portion of the transferred strand. However, in some preferredembodiments of the transposon end composition, the non-transferredstrand does not exhibit a sequence that is complementary to the5′-portion of the transferred strand. In some preferred embodiments, thenon-transferred strand exhibits only the non-transferred transposon endsequence. In some preferred embodiments, the non-transferred strandexhibits a sequence that is non-complementary to the transferred strand3′-of the non-transferred transposon end sequence.

In some embodiments of any of the methods comprising wherein thetransposon end composition comprises a transferred strand that comprisesor consists of a 5′-portion and a 3′-portion, the 5′-portion exhibitsthe sequence of a sequencing tag domain or a capture tag domain (e.g., asequencing tag domain or a capture tag domain for the Roche 454 GenomeSequencer FLX System, e.g., as the Roche 454A and 454B tags are used forsequencing using the Roche 454 Genome Sequencer FLX System) and the3′-portion exhibits the transferred transposon end sequence. Thus, whenthe transposon end composition comprises a transferred strand that has asequencing tag domain or a capture tag domain, the tagged circular ssDNAfragments or the di-tagged linear ssDNA fragments have a tag thatcomprises the sequencing tag domain or the capture tag domain (e.g., theRoche 454A or 454B tag used for sequencing using the Roche 454 GenomeSequencer FLX System). Tagged circular ssDNA fragments or the di-taggedlinear ssDNA fragments are generated which have the desired size rangeare used as templates for next-generation sequencing using the Roche 454Genome Sequencer FLX System. In other embodiments, the tagged circularssDNA fragments or the di-tagged linear ssDNA fragments are generatedthat comprise one or more restriction site domains, sequencing tagdomains, capture tag domains, amplification tag domains, detection tagdomains and/or address tag domains for use in sequencing (e.g., usingthe ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™ sequencingplatform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™ sequencingplatform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform, thePOLLONATOR Polony sequencing platform, the COMPLETE GENOMICS sequencingplatform, the INTELLIGENT BIOSYSTEMS' sequencing platform, or theHELICOS sequencing platform).

There is no limit to which additional sequences are used for the one ormore additional sequences in the 5′-portion of the transferred strand orin the 3′-portion of the non-transferred strand, which sequences can beused to accomplish any desired purpose. In some embodiments, the5′-portion of the transferred strand or the 3′-portion of thenon-transferred strand exhibits one or more tag domain sequences.

In some embodiments, the method further comprises the steps of extendingthe transferred strands comprising the 5′-tagged DNA fragments generatedin the transposition reaction using a DNA polymerase that lacksstrand-displacement and 5′-to-3′ exonuclease activity (e.g., T4 DNApolymerase, EPICENTRE), and then using a template-dependent DNA ligase(e.g., E. coli DNA ligase) to ligate the 3′-end of each DNA extensionproduct to the 5′-end of a non-transferred strand comprising the taggedDNA fragments using the opposite strand as a ligation template; in theseembodiments, the 5′-ends of the non-transferred strands of thetransposon end composition have a 5′-monophosphate group. Thisembodiment of the method generates di-tagged ssDNA fragments.

Work conducted during the development of embodiments of the presentinvention led to the observation that transposition occurs into dsDNA.Therefore, in some preferred embodiments, the transposon end compositioncomprises or consists of a non-transferred strand that exhibits only thenon-transferred transposon end sequence so that the 5′-portion of thetransferred strand is single-stranded (e.g., in order to minimize theprobability or frequency of insertion of the transferred strand intodouble-stranded portions of itself during the in vitro transpositionreaction). In some preferred embodiments wherein the non-transferredstrand exhibits a 3′-portion that is complementary to the 5′-portion ofthe transferred strand, the size of the 5′-portion of the transferredstrand is minimized in order to minimize the probability or frequency ofinsertion of the transferred strand into itself during the in vitrotransposition reaction. For example, in some embodiments, the size ofthe 5′-portion of the transferred strand (and of the complementary3′-portion of the non-transferred strand) is less than about 150nucleotides, less than about 100 nucleotides, less than about 75nucleotides, less than about 50 nucleotides, less than about 25nucleotides, or less than about 15 nucleotides.

In some preferred embodiments, the 5′-end of the transferred strand ofthe transposon end composition has a 5′-monophosphate group. In somepreferred embodiments, the 5′-end of the non-transferred strand has a5′-monophosphate group. In some preferred embodiments, both, thetransferred strand and the non-transferred strand have a5′-monophosphate group. In embodiments wherein the transferred stranddoes not have a 5′-monophosphate group, the method further comprises thestep of phosphorylating the 5′-end of the transferred transposon endoligonucleotide (e.g., using polynucleotide kinase; e.g., T4polynucleotide kinase) prior to the ligation step of the method.

Transposase-catalyzed insertion of the transposon end into the targetDNA results in joining of the transferred transposon end to the 5′-endof one strand of the target DNA and breakage or fragmentation of thatstrand at the site where the transferred transposon end sequence isjoined to the target DNA, with concomitant generation of a 9-base regionof single-stranded target DNA located 3′-of the site of joining of thetransferred transposon end to the target DNA due to a 9-base gap regionin the opposite strand of the target DNA. For example, FIG. 1 shows theresults of two independent insertion events of the transferredtransposon end into opposite strands of the target DNA. As shown in FIG.2, independent insertions of the transferred transposon end intoopposite strands of the target DNA sometimes occur at locations in thetarget DNA that are in relatively close proximity, generating two5′-tagged DNA fragments. Upon denaturation, two 5′-tagged ssDNAfragments are released.

Template-Independent Ligation of 5′-Tagged ssDNA Fragments

In some embodiments, a template-independent or non-homologous nucleicacid ligase (e.g., that carries out intramolecular ligation, i.e.,circularization of ssDNA that has a 3′-hydroxyl and a 5′-monophosphategroup) is used in a method of the invention for circularizing 5′-taggedlinear ssDNA fragments. In some preferred embodiments, the nucleic acidligase is a thermostable RNA ligase (e.g., selected from amongbacteriophage TS2126 thermostable RNA ligase (U.S. Pat. No. 7,303,901and Blondal et al., Nucleic Acids Res 33: 135-142, 2005), CIRCLIGASE™ssDNA ligase (EPICENTRE Biotechnologies, Madison, Wis., USA), and anarchael RNA ligase (e.g., Methanobacterium thermoautotrophicum RNAligase 1 or “MthRn1”; Torchia, C et al., Nucleic Acids Res.36:6218-6227, 2008). By a “template-independent ligase,” or“non-homologous ligase,” it is meant that a ligase that results inligation of ssDNA in the absence of annealing of a complementarysequence to the ends of the ssDNA that are to be joined or ligated(i.e., the two ends are not annealed to a complementary sequence inorder to keep them adjacent to each other during the ligation step). Inthese embodiments, the method comprises the step of: denaturing theannealed 5′-tagged DNA fragments generated in the in vitro transpositionreaction by incubating the 5′-tagged linear ssDNA fragments with thenucleic acid ligase. By “intramolecular ligation,” we mean that the twoends of one ssDNA molecule are ligated to each other to generatecircular ssDNA fragments, rather than being ligated to the ends of otherDNA molecules.

In some preferred embodiments, the non-homologous ligation reaction isperformed in an “improved ligation reaction mixture,” which herein meansa ligation reaction mixture that comprises: a) the tagged linear ssDNAfragments; b) a buffer that maintains the pH; b) Mn²⁺ cations; and (c) acomposition of thermostable RNA ligase molecules wherein a highproportion of the thermostable RNA ligase molecules are adenylated;wherein the concentration of the adenylated thermostable RNA ligasemolecules at least equals the molarity of the linear ssDNA fragments,and wherein no ATP or Mg²⁺ cations are added to the ligation reactionmixture.

By the statement that “a high proportion of the thermostable RNA ligasemolecules are adenylated”, it is meant that at least approximately 50%of all of the thermostable RNA ligase molecules in the improved ligationreaction mixture are adenylated. In some embodiments of the improvedligation reaction mixture, greater than approximately 60% of all of thethermostable RNA ligase molecules are adenylated. In some embodiments ofthe improved ligation reaction mixture, greater than approximately 70%of all of the thermostable RNA ligase molecules are adenylated. In someembodiments of the improved ligation reaction mixture, greater thanapproximately 80% of all of the thermostable RNA ligase molecules areadenylated. In some preferred embodiments of the improved ligationreaction mixture, greater than approximately 90% of all of thethermostable RNA ligase molecules are adenylated. In some preferredembodiments of the improved ligation reaction mixture, greater thanapproximately 95% of all of the thermostable RNA ligase molecules areadenylated. In some preferred embodiments, the thermostable RNA ligaseis adenylated in order to make a composition wherein a high proportionof the thermostable RNA ligase molecules are adenylated by incubatingthe enzyme with ATP during or after the purification process. Forexample, one protocol that can be used to adenylate the thermostable RNAligase is to incubate the enzyme in a solution containing 50 mMTris-HCl, pH 8.0, 2 mM MgCl₂, 100 mM NaCl, and 0.5 mM ATP for 15 minutesat 50 degrees C.; then stop the reaction by adding EDTA to a finalconcentration of 5 mM; and then to remove the reaction components bydialysis or gel filtration. The percent of adenylated thermostable RNAligase can be estimated by SDS-PAGE analysis. In some preferredembodiments, the thermostable RNA ligase wherein a high proportion ofthe thermostable RNA ligase molecules are adenylated is bacteriophageTS2126 thermostable RNA ligase. In some embodiments of the improvedligation reaction mixture, the buffer maintains the pH at between pH 6.5and 8.0. In some preferred embodiments of the improved ligation reactionmixture, the buffer maintains the pH at between pH 7.0 and 8.0. In somepreferred embodiments of the improved ligation reaction mixture, thebuffer that maintains the pH at between pH 7.0 and 8.0 is a Tris buffer.In some embodiments of the improved ligation reaction mixture, theconcentration of Mn²⁺ cations is between 0.5 and 10 mM. In someembodiments of the improved ligation reaction mixture, the concentrationof Mn²⁺ cations is between 1 and 10 mM. In some embodiments of theimproved ligation reaction mixture, the concentration of Mn²⁺ cations isbetween 1 and 5 mM. In some preferred embodiments of the improvedligation reaction mixture, the concentration of Mn²⁺ cations is 2.5 mM.In some preferred embodiments of the improved ligation reaction mixture,the Mn²⁺ cations are provided as MnCl₂. In some embodiments, theconcentration of the adenylated thermostable RNA ligase molecules in theimproved ligation reaction mixture is at least two-fold the molarity ofthe linear ssDNA fragments. In some embodiments, the concentration ofthe adenylated thermostable RNA ligase molecules in the improvedligation reaction mixture is at least five-fold the molarity of thelinear ssDNA fragments. In some embodiments, the concentration of theadenylated thermostable RNA ligase molecules in the improved ligationreaction mixture is at least ten-fold the molarity of the linear ssDNAfragments. In some preferred embodiments, the improved ligation reactionmixture additionally comprises a salt such as potassium chloride orpotassium acetate (e.g., at a concentration of about 50 to about 100mM). In some preferred embodiments, the improved ligation reactionmixture additionally comprises a reducing reagent such as dithiothreitol(DTT) (e.g., at a concentration of about 0.5 or 1 mM). In someembodiments, the improved ligation reaction mixture additionallycomprises zwitterionic trimethyl glycine (betaine) at a concentrationbetween 0.25 and 5.2 M. In some embodiments, the improved ligationreaction mixture additionally comprises zwitterionic trimethyl glycine(betaine) at a concentration between 0.5 and 2 M. In some embodiments,the improved ligation reaction mixture additionally compriseszwitterionic trimethyl glycine (betaine) at a concentration of about 1M.

In some preferred embodiments, the improved ligation reaction mixturecomprises: a) the linear ssDNA fragments that have 5′-phosphoryl and3′-hydroxyl groups (e.g., 0.5 micromolar); b) 33 mM TRIS acetate at pH7.8; b) 2.5 mM Mn²⁺ cations; and (c) a composition of thermostable RNAligase molecules wherein >70% of the thermostable RNA ligase moleculesare adenylated; wherein the concentration of the adenylated thermostableRNA ligase molecules at least equals the molarity of the linear ssDNAfragments (e.g., about 1 micromolar of adenylated thermostable RNAligase for 0.5 micromolar of the tagged linear ssDNA fragments), andwherein no ATP or Mg²⁺ cations are added to the ligation reactionmixture. In some preferred embodiments, the concentration of theadenylated thermostable RNA ligase molecules is at least 5 times, atleast 10 times, or at least 20 times the molarity of the linear ssDNAfragments (e.g., about 2.5 micromolar, about 5 micromolar, or about 10micromolar of adenylated thermostable RNA ligase for 0.5 micromolar ofthe tagged linear ssDNA fragments). In some preferred embodiments, theimproved ligation reaction mixture additionally comprises 66 mMpotassium acetate and 0.5 mM DTT. In some preferred embodiments, theimproved ligation reaction mixture additionally comprises 1 M betaine.

In some preferred embodiments of the method, intramolecular ligation of5′-tagged linear ssDNA fragments to synthesize tagged circular ssDNAfragments is performed in the ligation reaction mixture at a temperaturebetween about 40 degrees C. and about 70 degrees C. for sufficient time(e.g., from about one hour to about 72 hours) wherein tagged circularssDNA fragments are synthesized. In some preferred embodiments, theintramolecular ligation is performed at a reaction temperature of about60 degrees C. for sufficient time wherein circular ssDNA fragments aresynthesized.

The invention is not limited to only the particular nucleic acid ligasesdescribed herein. It will be understood by those with knowledge in theart that intramolecular ligation can be performed using any nucleic acidligase that has activity similar to those described herein, meaning thatit results in non-homologous intramolecular ligation of ssDNA that has a3′-hydroxyl and a 5′-monophosphate group.

V. Fragmentation and Tagging of Ds-DNA by In Vitro Transposition ofHairpin Transposon Ends (30963)

Briefly, in some embodiments, the method comprises: incubating thetarget DNA, which is dsDNA, with a transposase and a hairpin transposonend composition in an in vitro transposition reaction to simultaneouslyfragment and tag the target DNA, thereby generating a library comprisinga population of 5′-tagged DNA fragments; then joining the 3′ end of each5′-tagged DNA fragment, comprising a portion of one strand of target DNAto the 5′ end of another 5′-tagged DNA fragment comprising acomplementary portion (i.e., the opposite strand of the target DNA),thereby generating a library of covalently-closed tagged circular DNAfragments (e.g., that exhibit single-stranded circular- ordumbbell-shaped structures). In some preferred embodiments, the step ofjoining comprises: extending the 3′-ends of the 5′-tagged DNA fragmentswith a DNA polymerase that lacks 5′-to-3′ exonuclease (includingstructure-dependent 5′ nuclease) and strand displacement activities togenerate 5′-tagged DNA fragment extension products and ligating the 3′end of each of said 5′-tagged DNA fragment extension products to the 5′end of the complementary 5′-tagged DNA fragment extension product usinga template-dependent DNA ligase (e.g., E. coli DNA ligase or atemplate-dependent DNA ligase from a psychrophilic bacterium or apsychrophilic bacteriophage). In other preferred embodiments, the stepof joining comprises: incubating random-sequenceoligodeoxyribonucleotides (e.g., random-sequence or semi-random-sequenceoligodeoxyribonucleotides that are 5′-monophosphorylated or5′-adenylated) that are of one or more suitable sizes to exactly fillthe single-stranded gaps that results from the in vitro transpositionreaction (e.g., 5′-monophosphorylated random-sequenceoligodeoxyribonucleotides comprising or consisting of a random-sequenceor semi-random-sequence 9-mer or a random-sequence 4-mer and arandom-sequence 5-mer for filling the single-stranded gaps that resultsfrom the in vitro transposition using EZ-Tn5™ transposase) and atemplate-dependent DNA ligase (e.g., E. coli DNA ligase or atemplate-dependent DNA ligase from a psychrophilic bacterium or apsychrophilic bacteriophage) with the 5′-tagged DNA fragments underconditions and for sufficient time wherein the random-sequenceoligonucleotides anneal so as to the fill the single-stranded gaps inthe 5′-tagged DNA fragments and are ligated, thereby generating apopulation of tagged circular DNA molecules.

Thus, one preferred embodiment of the invention is a method forgenerating a library comprising a population of tagged circular DNAfragments from double-stranded target DNA for use as templates in DNAsequencing or nucleic acid amplification reactions, each of which taggedcircular DNA fragments exhibits the sequences of both strands of aportion of the target DNA and the sequence of the tag, the methodcomprising:

Providing:

1. target DNA comprising or consisting of one or more double-stranded(dsDNA) molecules (e.g., genomic, mitochondrial, chloroplast or otherdsDNA from a eukaryotic cell and/or genomic or episomal DNA from aprokaryotic cell, or double-stranded cDNA prepared by reversetranscription of RNA from a eukaryotic and/or prokaryotic cell togenerate first-strand cDNA and then extending a primer annealed to thefirst-strand cDNA);

2. a transposase (e.g., a wild-type or mutant transposase; e.g.,wild-type or mutant Tn5 transposase, e.g., EZ-Tn5™ transposase, e.g.,HYPERMU™ MuA transposase, EPICENTRE Biotechnologies, Madison, Wis.,USA); and

3. a hairpin transposon end composition that is capable of forming afunctional complex with the transposase in a transposition reaction andthat exhibits the sequence of the tag, wherein said hairpin transposonend composition comprises or consists of a 5′-phosphate-containingoligonucleotide that exhibits a non-transferred transposon end sequenceat its 5′-end (e.g., herein referred to as “MENTS” with respect to theEZ-Tn5™ non-transferred transposon end sequence), a transferredtransposon end sequence at its 3′-end (e.g., herein referred to as“METS” with respect to the EZ-Tn5™ transferred transposon end sequence),and an intervening sequence (e.g., for any desired purpose, such as toprovide a tag) between the non-transferred transposon end sequence andthe transferred transposon end sequence that is sufficiently long toallow intramolecular stem-loop formation, wherein the stem exhibits thesequences of the double-stranded transposon end with which thetransposase forms a complex that is functional for transposition (e.g.,wherein the stem exhibits the sequences of the 19-bp outer end (“OE”)transposon end, the 19-bp inner end (“IE”) transposon end, or the 19-bp“mosaic end” (“ME”) transposon end recognized by a wild-type or mutantTn5 transposase, e.g., by EZ-Tn5™ transposase) (or, e.g., R1 and R2MuAtransposon ends for MuA transposase) and the loop exhibits theintervening sequence, which can be an arbitrary sequence;

4. (a) a DNA polymerase that lacks 5′ nuclease (including 5′-to-3′exonuclease and structure-dependent 5′ nuclease activity) andstrand-displacement activities (e.g., T4 DNA polymerase); or (b) one ormore sizes of random-sequence oligonucleotides, which, alone, or incombination, have the same length as the single-stranded gaps in the5′-tagged DNA fragments that result following a transposition reactionwith the transposase and the hairpin transposon end composition; and

5. a template-dependent ligase (e.g., E. coli DNA ligase or atemplate-dependent ligase from a psychrophilic bacterium or apsychrophilic bacteriophage);

Incubating the target DNA in an in vitro transposition reaction with thetransposase and the hairpin transposon end composition under conditionsand for sufficient time wherein insertion of the hairpin transposon endcomposition into the target DNA generates a population of 5′-tagged DNAfragments (see, e.g., FIG. 2 and FIG. 3);

Incubating the 5′-tagged DNA fragments under conditions and forsufficient time wherein the single-stranded gaps in the DNA fragmentsare filled in and the 3′ end of each 5′-tagged DNA fragment is extendedand joined to the 5′-end of another 5′-tagged DNA fragment thatcomprises a complementary portion of the target DNA, thereby generatinga library of tagged circular DNA fragments, each of which exhibits thesequences of both strands of a portion of the target DNA and thesequence of the tag.

In some preferred embodiments of the method (as diagrammed, e.g., inFIGS. 7 and 8), the step of joining comprises: (1) incubating the5′-tagged DNA fragments with the DNA polymerase that lacks 5′ nucleaseactivity under conditions wherein the 3′-end of each 5′-tagged DNAfragment is extended to generate a population of 5′-tagged DNA fragmentextension products; and (2) incubating the 5′-tagged DNA fragmentextension products with the template-dependent ligase under conditionsand for sufficient time wherein the 5′-tagged DNA fragment extensionproducts are ligated, thereby generating the library of tagged circularDNA fragments. In some embodiments, the DNA polymerase that lacks 5′nuclease and strand-displacing activities and the template-dependentligase are provided in a mixture and the step of joining is carried outin a single reaction mixture.

In some other preferred embodiments of the method, the step of joiningcomprises: incubating the 5′-tagged DNA fragments with the one or moresizes of random-sequence oligonucleotides and the template-dependentligase under conditions and for sufficient time wherein therandom-sequence oligonucleotides anneal to and fill single-stranded gapregions in the 5′-tagged DNA fragments and wherein said annealedrandom-sequence oligonucleotides are ligated to each other or to anadjacent end of the 5′-tagged DNA fragments, thereby generating thetagged circular DNA fragments.

In some embodiments, the method additionally comprises, after theligation step, one or more steps to remove random-sequenceoligonucleotides, linear target DNA and/or the hairpin transposon endcompositions that are not joined to target DNA.

In some preferred embodiments, the method additionally comprises:treating the reaction mixture containing the tagged circular DNAfragments with T5 exonuclease (EPICENTRE Biotechnologies, Madison, Wis.)to remove unligated linear ssDNA and dsDNA (e.g., DNA fragments that arenicked and/or contain single-stranded regions).

In some embodiments, the method additionally comprises: cleaving thetagged circular DNA fragments in each of the loop structures to generatelinear double-stranded DNA fragments, each strand of which DNA fragmentshas a portion of the tag on its 5′-end and a portion of the tag on its3′-end (which linear DNA fragments are referred to herein as “fantaildi-tagged linear dsDNA fragments” or “fantail dsDNA fragments”).

In some embodiments, the method of cleaving comprises: annealing to thetagged circular DNA fragments an oligodeoxyribonucleotide that annealsto a restriction site within the tag, and then incubating with therestriction endonuclease that cleaves at the double-stranded restrictionsite to generate the fantail dsDNA fragments.

In some other embodiments, the hairpin transposon end composition hasone or more cleavable sites (e.g., in the loop structure) that arecleavable using a cleavage enzyme composition (e.g., a cleavable siteconsisting of a dUMP residue that is cleavable using a cleavage enzymecomposition comprising uracil-N-glycosylase and an AP endonuclease, suchas E. coli endonuclease III or endonuclease IV; or, e.g., a cleavablesite consisting of an 8-oxo-guanine-2′-deoxyribonucleoside-monophosphateresidue that is cleavable using a cleavage enzyme composition comprisingFPG protein±an AP endonuclease, such as E. coli endonuclease III orendonuclease IV), and the method of cleaving comprises: incubating withthe tagged circular DNA fragments with the cleavage enzyme compositionunder conditions and for sufficient time wherein the tagged circular DNAfragments are cleaved at the cleavable sites to generate the fantaildsDNA fragments. In some embodiments of the method, a differentnon-canonical nucleotide is used to provide the cleavable site and adifferent N-glycosylase is used in the cleavage enzyme composition. Thehairpin transposon end composition is synthesized (e.g., using anoligonucleotide synthesizer) to contain a cleavable site consisting of anon-canonical nucleotide in place of the canonical nucleotide (e.g.,dUMP as the non-canonical nucleotide in place of TMP whenuracil-N-glycosylase is used as a component of the cleaving enzymecomposition, or, e.g., 8-oxo-GMP as the non-canonical nucleotide inplace of GMP when FPG protein is used as a component of the cleavingenzyme composition) at the site or sites at which it is desired tocleave the tagged circular DNA fragments in the library (e.g., whereinthe site at which it is desired to cleave the tagged circular DNAfragments is within the loop structure of the hairpin transposon endcomposition that inserts into the tagged circular DNA fragments).

Thus, in some preferred embodiments wherein the transposon endcomposition has one or more cleavable sites, the cleavage enzymecomposition uses an N-glycosylase (or “DNA glycosylase”) to generate anabasic or apyrimidinic/apurinic (AP) site. As defined herein, an“N-glycosylase” is an enzyme that catalyzes hydrolysis of the bondbetween a non-canonical nucleic acid base and a sugar in DNA to generatean abasic (AP) site. Such enzymes are present in many species. Anexample from Escherichia coli is uracil N-glycosylase (UNG), also calleduracil-DNA glycosylase (UDG). UNG catalyzes the cleavage of the baseuracil from the sugar deoxyribose in DNA (Lindahl, Prog. Nucl. Acid Res.Mol. Biol. 22:135-192, 1979), but does not catalyze cleavage of uracilfrom free dUTP, free deoxyuridine or RNA (Duncan, in The Enzymes, Boyered., pp. 565-586, 1981). Other examples of N-glycosylases that can beused as cleavage enzymes are described by Demple and Harison (Annu Rev.Biochem. 63: 915-48, 1994) and by Duncan (“DNA Glycosylases,” in TheEnzymes, Boyer ed., p. 565-586, 1981). By “N-glycosylase” or“DNA-glycosylase” we mean an enzyme with N-glycosylase activity, whetheror not the enzyme is formally called a glycosylase or has a glycosylaseactivity combined with other enzymatic activities. Glycosylases aresometimes referred to as “glycosidases,” and we therefore mean thedefinition of N-glycosylase to cover N-glycosidases. For example, FPGprotein is also an N-glycosylase as defined herein. FPG protein(Formamidopyrimidine DNA N-glycosylase) is a base excision repair enzymethat recognizes diverse but structurally related modified nucleic acidbases such as 8-hydroxyguanine (also known as 7-hydro-8-oxoguanine or8-oxoguanine, referring to the favored 6,8-diketo tautomer atphysiological pH) (Tchou, et al., Proc. Natl. Acad. Sci. USA 88:4690-4694, 1991), imidazole ring-opened derivatives of adenine orguanine, designated 4,6-diamino-5-formamidopyrimidine and2,6-diamino-4-hydroxy-5-formamidopyrimidine, respectively, (Chetsanga,et al., Biochemistry 20: 5201-5207, 1981; and Breimer, Nucl. Acids Res.12: 6359-6367, 1984), N.sub.7-methylformamidopyrimidines,5-hydroxyuracil and 5-hydroxycytosine (Hatahet, et al., J. Biol. Chem.269:18814-18820, 1994) and catalyzes the cleavage of the N-glycosyllinkage between the modified base and the deoxyribose-phosphodiesterbackbone in DNA, generating an AP site. In addition, FPG protein alsopossesses an AP lyase activity. The AP-lyase activity of the enzymecatalyzes beta, delta-elimination reactions, leaving a single-nucleotidegap in the DNA (Bailly, et al., Biochem. J. 261: 707-713, 1989). FPGprotein and 8-hydroxyguanine DNA glycosylase have been shown to beidentical (Chung, M H et al., Mutation Research 254: 1-12, 1991).Treatment of DNA with methylene blue plus visible light (Floyd, et al.,Arch Biohem Biophys 273: 106-111, 1989) or with rose bengal in plusultraviolet light (Friedmann and Brown, Nucleic Acids Research 5:615-622, 1978) induces guanine-specific modification that is cleavableby FPG protein. Other specific N-glycosylases will be available andknown to those of skill in the art. In order to determine whether or notan N-glycosylase is suitable for the present invention, one would firstincorporate the non-canonical nucleotide into the DNA and determinewhether or not the non-canonical base can be specifically removed by thecandidate N-glycosylase in a manner similar to removal of uracil or8-oxo-guanine by UNG and FPG protein, respectively. Once one has createdan abasic or AP site, various methods are known in the art to cleave theabasic site. Heat and/or basic conditions may be used to break the DNAmolecule at the abasic sites. For example, the following protocol may beused: Nucleic acids containing abasic (AP) sites following removal ofnon-canonical bases are heated in a buffer solution containing an amine,for example, 25 mM Tris-HCl and 1 to 5 mM magnesium ions, for a periodof 10 to 30 minutes at 70 degrees C. to 95 degrees C. Alternatively, thefollowing treatment may be used to break the DNA at abasic sites: 1.0 Mpiperidine, a base, is added to DNA which has been precipitated withethanol and vacuum dried. The solution is then heated for 30 minutes at90 degrees C. and lyophilized to remove the piperidine. In somepreferred embodiments, enzymatic treatment using anapurinic/apyrimidinic endonuclease (AP endonuclease) known in the art(Lindahl, Prog. Nucl. Acid Res. Mol. Biol. 22: 135-192, 1979; Demple andHarison Annu Rev. Biochem. 63: 915-48, 1994) is used to break the DNApolymer at the abasic site. As defined herein, an AP endonuclease is anyenzyme that catalyzes cleavage of DNA at abasic (AP) sites. Such enzymesare present in many species. Examples of AP endonucleases from E. coliinclude, but are not limited by, endonuclease III and endonuclease IV.Also, E. coli exonuclease III in the presence of calcium ions is an APendonuclease. Enzymes useful in the present invention include any enzymewith AP endonuclease-like activity, whether it is called by that name orby some other name.

In some preferred embodiments, the method additionally comprises:denaturing the fantail dsDNA fragments to generate a library ofdi-tagged linear ssDNA fragments (e.g., for use as templates for DNAsequencing or DNA amplification).

In some embodiments the tagged circular DNA fragments or the di-taggedlinear ssDNA fragments in the library generated using the methods areused as DNA templates in nucleic acid amplification and/or DNAsequencing reactions. In some embodiments, the method further comprisesthe step of amplifying and/or sequencing the target DNA in the taggedcircular DNA fragments or the di-tagged linear ssDNA fragments. In someembodiments, the method further comprises the step of sequencing DNAthat is complementary to the target DNA obtained by amplification of thetagged circular DNA fragments or the di-tagged linear ssDNA fragments.In some embodiments, at least a portion of the target DNAs in each ofthe tagged circular DNA fragments or the di-tagged linear ssDNAfragments is sequenced using a DNA polymerase and at least one primerthat is complementary to the tag (e.g., for sequencing by synthesis). Insome embodiments, at least a portion of the target DNAs in each of thetagged circular DNA fragments or the di-tagged linear ssDNA fragments issequenced using a template-dependent ligase to ligate at least oneoligodeoxyribonucleotide that is complementary to the tag and at leastone other oligodeoxyribonucleotide that anneals to the portion of thetarget sequence (e.g., for sequencing by ligation). In some embodiments,at least a portion of the target DNA in each of the tagged circular DNAfragments or the di-tagged linear ssDNA fragments is sequenced byannealing oligodeoxyribonucleotides that anneal or hybridize to the tagand to a portion of the target sequence (e.g., for sequencing byhybridization). In some embodiments, DNA that is complementary to thetagged circular DNA fragments or the di-tagged linear ssDNA fragments issequenced using sequencing by synthesis, sequencing by ligation, orsequencing by hybridization.

For example, in some preferred embodiments, the transferred transposonend sequence exhibited by the hairpin transposon end composition that isprovided in a kit or that is used in a method of the present inventionis a transferred transposon end sequence recognized by a Tn5transposase. In some preferred embodiments, the transferred transposonend sequence is a sequence recognized by EZ-Tn5™ transposase (EPICENTREBiotechnologies, Madison, Wis., USA).

In general, the tagged circular DNA fragments, the fantail dsDNAfragments, and the di-tagged linear ssDNA fragments generated using ahairpin transposon end composition in the methods of the presentinvention exhibit both the transferred transposon end sequence and thenon-transferred transposon end sequence, and additional sequencescomprising or derived from the non-complementary loop portion of thehairpin transposon end composition. Thus, in some embodiments, thehairpin transposon end composition exhibits one or more other nucleotidesequences 5′-of the transferred transposon end sequence and 3′-of thenon-transferred transposon end sequence, which one or more othernucleotide sequences are also exhibited by the tag. Thus, in addition tothe transposon end sequences, the tag of the hairpin transposon endcomposition can have one or more other tag portions or tag domains.

In some embodiments wherein the hairpin transposon end compositioncomprises one or more restriction site domains, the method furthercomprises: annealing an oligodeoxyribonucleotide that is complementaryto the single-stranded restriction site of the tagged circular DNAfragments and then cleaving the tagged circular DNA fragments at therestriction site using the restriction endonuclease that recognizes therestriction site. Thus, in some embodiments, the method compriseslinearizing the tagged circular DNA fragments to generate fantail dsDNAfragments or, following denaturation, di-tagged linear ssDNA fragments.

In some embodiments, the method further comprises the step of ligatingthe restriction endonuclease-cleaved tagged linear ssDNA fragments toone or more other DNA molecules (e.g., for joining a tag).

Thus, in some embodiments, the method further comprises: amplifying thetagged circular DNA fragments or the fantail dsDNA fragments or thedi-tagged linear ssDNA fragments by transcription, the methodcomprising: (a) annealing to the sense promoter sequence anoligodeoxyribonucleotide that exhibits a complementary anti-sensepromoter sequence, or, annealing to the tagged circular DNA fragments orthe fantail dsDNA fragments or the di-tagged linear ssDNA fragments aprimer that is complementary thereto and extending the primer with a DNApolymerase under conditions wherein a dsDNA, including a double-strandedRNA polymerase promoter, is synthesized; and (b) incubating the dsDNAproducts with an RNA polymerase that binds the RNA polymerase promoterunder conditions wherein RNA is synthesized.

In some preferred embodiments wherein the hairpin transposon endcomposition or a PCR primer exhibits an RNA polymerase promotersequence, the RNA polymerase promoter is a T7-type RNA polymerasepromoter and the method further comprises the step of transcribing thetagged circular DNA fragments in vitro using a T7-type RNA polymerasethat recognizes the promoter. Most preferably, the RNA polymerase andpromoter are chosen from among T7 RNAP, T3 RNAP and SP6 RNAP and thecorresponding cognate promoters. However, transcription steps of amethod of the invention can use any RNAP for which a suitable promotersequence that permits transcription with high specificity is known orcan be obtained. Kits and enzymes for in vitro transcription arecommercially available from many vendors and the appropriate reactionmixtures and conditions for carrying out steps of the present inventioncomprising in vitro transcription can use those products as described bythe manufacturers. By way of example but in vitro transcription using T7RNAP can be carried out using the AMPLISCRIBE™ T7-FLASH™ TranscriptionKit or the AMPLISCRIBE™ T7 High Yield Transcription Kit from EPICENTREBiotechnologies, Madison, Wis. as described in the product literature.Similarly, if T3 RNAP or SP6 RNAP is used in a method of the inventionfor in vitro transcription, an AMPLISCRIBE™ T3-FLASH™ High YieldTranscription Kit or with the AMPLISCRIBE™ SP6 High Yield TranscriptionKit (EPICENTRE Biotechnologies, Madison, Wis.), respectively, can beused as described.

In some other embodiments, the method further comprises the step ofamplifying and/or sequencing the target DNA in the tagged circular DNAfragments using a DNA polymerase and at least one primer that iscomplementary to the tag. In some embodiments, the method additionallycomprises: amplifying the tagged circular DNA fragments by rollingcircle replication using a strand-displacing DNA polymerase. In someother embodiments, the method additionally comprises: amplifying thetagged circular DNA fragments by PCR using a thermostable DNApolymerase, a first PCR primer that is complementary to at least aportion of the tag, and a second PCR primer that is complementary to atleast a portion of the complement of the tag.

In some embodiments, the method further comprises: amplifying the taggedcircular DNA fragments by rolling circle replication (RCR), the methodcomprising: (a) annealing a primer that is complementary to the taggedcircular DNA fragments; and (b) extending the primer annealed to thetagged circular DNA fragments using a strand-displacing DNA polymerase(e.g., phi29 DNA polymerase, rBst DNA polymerase large fragment orDISPLACEACE™ DNA polymerase (EPICENTRE). In these embodiments, the RCRamplification products are concatameric ssDNA molecules that arecomplementary to the tagged circular DNA fragments. In some embodimentswherein the tagged circular DNA fragments exhibit an anti-sense promotersequence, the concatameric RCR amplification products exhibit a sensepromoter sequence and the method further comprises making the RNApolymerase promoter double-stranded (e.g., by annealing to the sensepromoter sequence a complementary oligodeoxyribonucleotide that exhibitsan anti-sense promoter sequence, and then transcribing the concatamericDNA using an RNA polymerase that binds to the double-stranded RNApolymerase promoter and initiates transcription therefrom.

In some preferred embodiments, the stem portion of the hairpintransposon end composition exhibits only the transferred and thenon-transferred transposon end sequences and the loop is single-stranded(e.g., in order to minimize the probability or frequency of insertion ofthe hairpin transposon end composition into double-stranded portions ofitself during the in vitro transposition reaction). In some otherembodiments, the stem portion of the hairpin transposon end compositionexhibits, in addition to the transferred and non-transferred transposonend sequences, additional sequences that are immediately 5′-of thetransferred transposon end sequence and immediately 3′-of thenon-transferred transposon end sequence. However, in these embodiments,the size of the additional sequences in the stem portion of the hairpintransposon end composition is minimized in order to minimize theprobability or frequency of insertion of the hairpin transposon endcomposition into itself during the in vitro transposition reaction. Forexample, in some embodiments, the length of the stem in the hairpintransposon end composition is less than about 75 nucleotides; less thanabout 50 nucleotides; or less than about 30 nucleotides.

In some embodiments, the loop portion of the hairpin transposon endcomposition exhibits the sequence of a sequencing tag domain or acapture tag domain (e.g., a sequencing tag domain and/or a capture tagdomain for the Roche 454 Genome Sequencer FLX System, e.g., that exhibitthe sequences of the sequencing tag domains of the Roche 454A and 454Btags that are used for sequencing using the Roche 454 Genome SequencerFLX System). In some embodiments wherein the hairpin transposon endcomposition has a sequencing tag domain or a capture tag domain, thetagged circular DNA fragments or the fantail dsDNA fragments or thedi-tagged linear ssDNA fragments have a tag that comprises thesequencing tag domain and/or the capture tag domain (e.g., the Roche454A or 454B tag used for sequencing using the Roche 454 GenomeSequencer FLX System). After isolating the tagged circular DNA fragmentsor the fantail dsDNA fragments or the di-tagged linear ssDNA fragmentsin the desired size range, they are used as templates fornext-generation sequencing using the Roche 454 Genome Sequencer FLXSystem. In other embodiments, the tagged circular DNA fragmentsgenerated or the fantail dsDNA fragments or the di-tagged linear ssDNAfragments have one or more restriction site domains, sequencing tagdomains, amplification tag domains, capture tag domains, detection tagdomains and/or address tag domains for use in sequencing (e.g., usingthe ROCHE 454 sequencing platform, the ILLUMINA™ SOLEXA™ sequencingplatform, the LIFE TECHNOLOGIES/APPLIED BIOSYSTEMS' SOLID™ sequencingplatform, the PACIFIC BIOSCIENCES' SMRT™ sequencing platform, thePOLLONATOR Polony sequencing platform, the COMPLETE GENOMICS sequencingplatform, the INTELLIGENT BIOSYSTEMS' sequencing platform, or theHELICOS sequencing platform).

In some embodiments, the hairpin transposon end composition exhibits oneor more tag domain sequences, which sequences can be used to accomplishany desired purpose. There is no limit to which additional sequences areused for the one or more additional sequences in the loop portion of thehairpin transposon end composition.

In some preferred embodiments, the 5′-end of the hairpin transposon endcomposition has a 5′-monophosphate group. In embodiments wherein thehairpin transposon end composition does not have a 5′-monophosphategroup, the method further comprises the step of phosphorylating the5′-end of the hairpin transposon end composition (e.g., usingpolynucleotide kinase; e.g., T4 polynucleotide kinase) prior to theligation step of the method.

Transposase-catalyzed insertion of the transposon end into the targetresults in joining of the 3′ end of the transferred transposon endsequence to the 5′ position of a nucleotide in one strand of the targetDNA, resulting in breakage or fragmentation of that strand at the sitewhere the transferred transposon end sequence is joined to the targetDNA and concomitant generation of a 9-base region of single-strandedtarget DNA located 3′-of the site of joining of the transferredtransposon end to the target DNA due to a 9-base gap region in theopposite strand of the target DNA. For example, FIG. 16 shows onepossible result of two independent insertion events of the hairpintransposon end composition into the target DNA. As shown in FIG. 16,independent insertions of the hairpin transposon end into oppositestrands of the target DNA sometimes occur at locations in the targetDNA, generating two 5′-tagged DNA fragments as shown in FIG. 16. Uponjoining, a tagged circular DNA fragment is generated.

The invention is not limited to only the particular nucleic acid ligasesdescribed herein. It will be understood by those with knowledge in theart that any template-dependent nucleic acid ligase that has activitysimilar to the enzymes described herein, and methods and conditions forusing of such enzymes for template-dependent ligation are known andreadily available in the art.

EXPERIMENTAL EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions.

Standard molecular biology techniques used are well known in the art andare described by Sambrook, J., Fritsch, E. F. and Maniatis, T.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Definitions, Nomenclature and Abbreviations used in the Examples:

“pMETS” refers to the 19-base 5′-phosphate-containing single-strandedtransposon end oligonucleotide that exhibits the EZ-Tn5™ transposon endsequence:

5′ pAGA TGT GTA TAA GAG ACAG 3′ (SEQ ID NO: 1)

“METS” refers to the 19-base single-stranded transposon endoligonucleotide that exhibits the EZ-Tn5™ transposon end sequence:

5′ AGA TGT GTA TAA GAG ACAG 3′ (SEQ ID NO: 1)

“pMENTS” refers to the 19-base 5′-phosphate-containing single-strandedtransposon end oligonucleotide that exhibits the EZ-Tn5™ transposon endsequence:

5′ pCTG TCT CTT ATA CAC ATCT 3′ (SEQ ID NO: 2)

“pMEDS” refers to the 19-basepair double-stranded EZ-Tn5™ transposon endwherein both 5′-ends contain phosphates:

5′ pAGA TGT GTA TAA GAG ACAG 3′ (SEQ ID NO: 1) 3′  TCT ACA CAT ATT CTCTGTCp 5′ (SEQ ID NO:2)

The pMEDS EZ-Tn5™ transposon end is made by annealing the pMETStransposon end oligonucleotide to the pMENTS transposon endoligonucleotide.

“MEDS” refers to the 19-basepair double-stranded EZ-Tn5™ transposon endwherein only the non-transferred strand (pMENTS) contains a5′-phosphate:

5′ AGA TGT GTA TAA GAG ACAG 3′ (SEQ ID NO: 1) 3′ TCT ACA CAT ATT CTCTGTCp 5′ (SEQ ID NO:2)

The MEDS EZ-Tn5™ transposon end is made by annealing the METS transposonend oligonucleotide to the pMENTS transposon end oligonucleotide.

“p454.1METS” refers to the 36-base 5′-phosphate-containingsingle-stranded transferred strand that has a 5′-portion consisting of aRoche 454 sequencing tag that exhibits the sequence below, which isappended to the 5′ end of the underlined 19-base EZ-Tn5™ transferredtransposon end sequence (pMETS):

(SEQ ID NO: 4) 5′ pGCC TTG CCA GCC CGC TCA GAT GTG TAT AAG AGA CAG 3′

“p454.1MEDS” EZ-Tn5™ transposon end composition is made by annealing thep454.1METS transferred strand (SEQ ID NO:4) to the pMENTSnon-transferred strand (SEQ ID NO:2):

5′ pGCC TTG CCA GCC CGC TCA GAT GTG TAT AAG AGA                        3′T CTA CAC ATA TTC TCT CAG 3′ GTCp 5′

“pc454.1” refers to the 18-base 5′-phosphate-containing single-strandedoligonucleotide that is complementary the 5′ portion of p454.1METS andhas the sequence:

5′ pTGA GCG GGC TGG CAA GGC 3′ (SEQ ID NO: 5)

“A-METS” refers to the 38-base single-stranded transferred strand thathas a 5′-portion consisting of a Roche 454 sequencing tag that exhibitsthe sequence below, which is appended to the 5′ end of the underlined19-base EZ-Tn5™ transferred transposon end sequence (METS):

(SEQ ID NO: 7) 5′GCC TCC CTC GCG CCA TCA GAG ATG TGT ATA AGA GAC AG 3′

“A-MEDS” EZ-Tn5™ transposon end composition is made by annealing theA-METS transferred strand (SEQ ID NO:7) to the pMENTS non-transferredstrand (SEQ ID NO:2):

5′ GCC TCC CTC GCG CCA TCA GAG ATG TGT ATA AGA GAC                          3′TC TAC ACA TAT TCT CTG AG 3′ TCp 5′

“B-METS” refers to the 38-base single-stranded transferred strand thathas a 5′-portion consisting of a Roche 454 sequencing tag that exhibitsthe sequence below, which is appended to the 5′ end of the underlined19-base EZ-Tn5™ transferred transposon end sequence (METS):

(SEQ ID NO: 8) 5′ GCC TTG CCA GCC CGC TCA GAG ATG TGT ATA AGA GAC AG 3′

“B-MEDS” EZ-Tn5™ transposon end composition is made by annealing theB-METS transferred strand (SEQ ID NO:8) to the pMENTS non-transferredstrand (SEQ ID NO:2)

5′ GCC TTG CCA GCC CGC TCA GAG ATG TGT ATA AGA GAC                          3′TC TAC ACA TAT TCT CTG AG 3′ TCp 5′

“FLX-A” refers to the 19-base single-stranded oligonucleotide thatconsists of a Roche 454 sequencing tag that exhibits the sequence below:

5′ GCC TCC CTC GCG CCA TCA G 3′ (SEQ ID NO: 9)

“FLX-B” refers to the 19-base single-stranded oligonucleotide thatconsists of a Roche 454 sequencing tag that exhibits the sequence below:

5′ GCC TTG CCA GCC CGC TCA G 3′ (SEQ ID NO: 10)

“A-MID2-METS” refers to the 48-base single-stranded transferred strandthat has a 5′-portion consisting of a Roche 454 sequencing tag and barcode sequence (MID2, italics) that exhibits the sequence below, which isappended to the 5′ end of the underlined 19-base EZ-Tn5™ transferredtransposon end sequence (METS):

(SEQ ID NO: 11) 5′ GCC TCC CTC GCG CCA TCA G ACGCTCGACA AG ATG TGTATA AGA GAC AG 3′

“Ti A-METS” refers to the 49-base single-stranded transferred strandthat has a 5′-portion consisting of a Roche 454 sequencing tag thatexhibits the sequence below, which is appended to the 5′ end of theunderlined 19-base EZ-Tn5™ transferred transposon end sequence (METS):

(SEQ ID NO: 12) 5′ CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG AGA TGTGTA TAA GAG ACA G 3′

“Ti B-METS” refers to the 49-base single-stranded transferred strandthat has a 5′-portion consisting of a Roche 454 sequencing tag thatexhibits the sequence below, which is appended to the 5′ end of theunderlined 19-base EZ-Tn5™ transferred transposon end sequence (METS):

(SEQ ID NO: 13) 5′ CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG AGA TGTGTA TAA GAG ACA G 3′

“Ti A” refers to the 26-base single-stranded oligonucleotide thatconsists of a Roche 454 sequencing tag that exhibits the sequence below:

(SEQ ID NO: 14) 5′ CCA TCT CAT CCC TGC GTG TCT CCG AC 3′

“Ti B” refers to the 26-base single-stranded oligonucleotide thatconsists of a Roche 454 sequencing tag that exhibits the sequence below:

(SEQ ID NO: 15) 5′ CCT ATC CCC TGT GTG CCT TGG CAG TC 3′

“BP1-A” refers to the 48-base single-stranded oligonucleotide that has a5′-portion consisting of an Illumina bridge PCR tag that exhibits thesequence below, which is appended to the FLX-A sequence (underlinedbelow):

(SEQ ID NO: 16) 5′ AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG CCT CCCTCG CGC CAT CAG 3′

“BP2-B” refers to the 49-base single-stranded oligonucleotide that has a5′-portion consisting of an Illumina bridge PCR tag that exhibits thesequence below, which is appended to the FLX-B sequence (underlinedbelow):

(SEQ ID NO: 17) 5′ CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT GCC TTGCCA GCC CGC TCA G 3′

“BP2-ID1-B” refers to the 49-base single-stranded oligonucleotide thathas a 5′-portion consisting of an Illumina bridge PCR tag and bar codesequence (ID2, italics) that exhibits the sequence below, which isappended to the FLX-B sequence (underlined below):

(SEQ ID NO: 18) 5′ CAA GCA GAA GAC GGC ATA CGA GAT GCATGT CGG TCTGCC TTG CCA GCC CGC TCA G 3′

“BP1” refers to the 20-base single-stranded oligonucleotide thatconsists of an Illumina bPCR adaptor tag that exhibits the sequencebelow:

5′ AAT GAT ACG GCG ACC ACC GA 3′ (SEQ ID NO: 19)

“BP2” refers to the 21-base single-stranded oligonucleotide thatconsists of an Illumina bPCR adaptor tag that exhibits the sequencebelow:

5′ CAA GCA GAA GAC GGC ATA CGA 3′ (SEQ ID NO: 20)

“pMETS-N-MENTS” refers to a hairpin transposon end compositioncomprising or consisting of: a 5′-phosphate-containing oligonucleotidethat exhibits the EZ-Tn5™ non-transferred transposon end sequence at the5′-end and the EZ-Tn5™ transferred transposon end sequence at the3′-end, connected by an intervening arbitrary sequence represented by“(N)x”. The intervening sequence between the METS and MENTS sequencesconsists of a sufficient number of nucleotides to allow stem-loopformation:

(SEQ ID NO: 3) 5′PCTGTCTCTTATACACATCT-(N)_(x)-AGATGTGTATAAGAGACAG 3′

Intramolecular annealing of pMETS transferred transposon end sequence tothe pMENTS non-transferred transposon end sequence within apMETS-N-MENTS oligonucleotide makes a Hairpin EZ-Tn5™ transposon endcomposition. For example, if x=6;

 NN (SEQ ID NO: 3) N  AGATGTGTATAAGAGACAG  3′ N  TCTACACATATTCTCTGTCp 5′ NN

“TSase” refers to the hyperactive EZ-Tn5™ Tn5 transposase (EPICENTREBiotechnologies, Madison, Wis., USA) in 50 mM Tris chloride pH 7.5, 50%glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM Sodium chloride, 0.5% v/v NP-40,0.5% v/v Tween-20.

“Transposome” refers to the hyperactive EZ-Tn5™ Tn5 transposase(EPICENTRE Biotechnologies, Madison, Wis., USA) preincubated withdouble-stranded transposon DNA under conditions that supportnon-covalent complex formation. Double-stranded transposon DNA canconsist of, without limitation, Tn5 DNA, a portion of Tn5 DNA, atransposon end composition, a mixture of transposon end compositions orother double-stranded DNAs capable of interacting with the hyperactiveEZ-Tn5™ transposase.

10X TA Reaction Buffer: 330 mM Tris acetate, pH 7.8 100 mM Magnesiumacetate 660 mM potassium acetate 5X TA-DMF Reaction Buffer: 165 mM Trisacetate, pH 7.8 50 mM Magnesium acetate 330 mM potassium acetate 50% v/vdimethylformamide 10X TMgCl Reaction Buffer: 100 mM Tris chloride, pH8.0 50 mM Magnesium chloride 5X TMgCl-DMF Reaction Buffer: 50 mM Trischloride, pH 8.0 25 mM Magnesium chloride 50% v/v dimethylformamide 10XTMgAc Reaction Buffer: 100 mM Tris acetate, pH 7.6 50 mM Magnesiumchloride 5X TMgAc-DMF Reaction Buffer: 50 mM Tris acetate, pH 7.6 25 mMMagnesium chloride 50% v/v dimethylformamide

“Target DNA” refers to the DNA subjected to transposition. In theexample below, bacteriophage T7D111 DNA is used as the target DNA.

“TSase” refers to the hyperactive EZ-Tn5™ Tn5 transposase (EPICENTREBiotechnologies, Madison, Wis., USA).

10× Transposase Reaction Buffer:

-   -   330 mM Tris acetate, pH 7.8    -   100 mM Magnesium acetate    -   660 mM potassium acetate

Example 1 In Vitro Transposition-Mediated DNA Fragmentation and5′-Tagging Using EZ-Tn5™ Transposase and the EZ-Tn5™ Transposon End

The following reaction mixture was assembled:

x water to a final volume of 50 microliters 5 microliters 10X EZ-Tn5 ™Transposition Buffer 1 microgram target DNA in 1 to 40 microliters 2microliters pMEDS (25 micromolar)* 2 microliters EZ-Tn5 ™ Transposase(at 10 units per microliter) 50 microliters *In some embodiments, twodifferent pMEDS transposon ends that each additionally exhibits adifferent arbitrary sequence in its respective 5′-portion of thetransferred transposon end, 5′-of the transferred transposon endsequence (FIG. 4).

After mixing, the reaction was incubated for 1 hour at 37° C. Thereaction was stopped with 10 microliters of stop solution (15% sucrose,66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma 0-7252],and Proteinase K at 100 micrograms per ml), mixed, and heated at 50° C.for 10 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. LMPagarose was used to isolate DNA into size classes. Gels were stainedwith SYBR Gold and DNA was visualized with non-UV light. Gel slices forLMP gels were incubated at 70° C. for 5 minutes to liquify the gel.After 5 minutes at 37° C., one-hundredth volume of Gelase™ agarosedigesting solution (EPICENTRE Biotechnologies) was added. The reactionwas mixed and was incubated for 1 hour at 37° C.

The target DNA was fragmented to a similar extent and to a similar sizerange as described in Examples 3 and 4 using comparable quantities andconcentrations of the EZ-Tn5™ Tn 5 transposase and the transposon ends.DNA from the sizing procedure was used in EXAMPLE 2 for tagging the 3′ends of the 5′-tagged DNA fragments.

Example 2 Size Range of 5′-Tagged DNA Fragment Transposition ProductsUsing Different EZ-Tn5™ Tn5 Transposase Concentrations

Tn5 hyperactive EZ-Tn5™ transposase (EPICENTRE) at a concentration of 90units per microliter was diluted to final concentrations of 45, 22.5,11.3 and 9 units per microliter. Two microliters of the enzyme at eachconcentration were incubated with 1 microgram of phage T7 D111 targetDNA (having a size of about 39 Kbp) and 1 micromolar of the pMEDStransposon end in TA buffer in a final reaction mixture volume of 50microliters for 1 hour at 37° C.

The reactions were stopped with 10 microliters of a stop solutioncontaining 15% sucrose, 66 mM EDTA, 20 mM Tris/HCl pH 8.0, 0.1% SDS,0.9% orange G and 100 micrograms per ml of proteinase K. After mixingand incubation at 50° C. for 10 min, 10-microliter aliquots wereelectrophoresed on a 1% agarose gel in TAE buffer for 1 hour at 100volts. The gel was stained with SYBR Gold and photo-graphed with A340transillumination.

A final concentration of about 0.9 unit per microliter of Tn5transposase in the reaction mixture gave maximal fragmentation of thephage T7 D111 target DNA. Higher concentrations of the Tn5 transposasewere inhibitory and lower concentrations shifted the fragment size rangeupward. At a final concentration of about 0.9 unit of Tn5 transposaseper microliter, the majority of the phage T7 D111 target DNA wasfragmented into DNA that migrated on the gel at sizes between about 150by and about 1.5 Kbp based on the marker bands. At a final concentrationof about 0.45 unit of Tn5 transposase per microliter, the majority ofthe phage T7 D111 target DNA was fragmented into DNA that migrated onthe gel at sizes between about 400 by and about 3.5 Kbp based on themarker bands.

Example 3 Size Range of 5′-Tagged DNA Fragment Transposition ProductsUsing Different pMEDS Transposon End Concentrations

A 25-micromolar stock of the pMEDS transposon end was serially diluted2-, 4-, and 8-fold with T₁₀E₁ buffer. Then, 2 microliters of eachtransposon end dilution and a no-transposon-end buffer control wereincubated in 50-microliter reactions containing 1×TA buffer, 1 microgramof phage 7 D111 target DNA, and 0.4 units per microliter of hyperactiveTn5 transposase for 1 hour at 37° C.

The reactions were stopped and samples analyzed by 1% agarose gelelectrophoresis as described in EXAMPLE 2.

The 4-fold dilution of the 25 uM stock, which resulted a finalconcentration of 0.25 micromolar of the pMEDS transposon end in thereaction mixture, resulted in good fragmentation of the target DNA, andwas probably most efficient in terms of use of the pMEDS transposon end.At this concentration, the majority of the phage T7 D111 target DNA wasfragmented into DNA that migrated on the gel at sizes between about 400by and about 3.5 Kbp based on the marker bands. At 0.5 and 1 micromolarconcentrations of the pMEDS transposon end, the sizes of the fragmentedDNA were shifted downward slightly to between about 200-300 by and about3 Kbp.

Example 4 Size Range of 5′-Tagged DNA Fragment Transposition ProductsUsing Different Transposome Concentrations

“A-MEDS Transposomes” and “B-MEDS Transposomes” were formed bypre-incubating 12.5 μM TSase with 12.5 μM A-MEDS or 12.5 μM B-MEDStransposon end compositions, respectively, for 60 minutes at 37° C.A-MEDS and B-MEDS transposomes were combined in equal ratios to form“A/B Transposomes.”

Transposomes were then used at 12.5 μM, or diluted to 10 μM, 7.5 μM, 5μM, 2.5 μM, or 1 μM with TSase storage buffer (50 mM Tris chloride pH7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM Sodium chloride, 0.5%v/v NP-40, 0.5% v/v Tween-20). E. coli genomic DNA was 5′-end tagged andfragmented using A/B Transposomes in the following reactions:

Reagent Volume 5X TMgCl-DMF Reaction Buffer 4 μl 50 ng/μl E. coligenomic DNA 1 μl A/B Transposome (12.5, 10, 7.5, 5, 2.5, or 1 μM) 1 μlwater 14 μl Final Volume: 20 μl

The reactions were incubated 5 minutes at 55° C. Then, the reactionswere stopped with 5 microliters of stop solution (15% sucrose, 66 mMEDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma 0-7252], andProteinase K at 100 micrograms per ml), mixed, and heated at 70° C. for10 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. Gelswere stained with SYBR Gold and DNA was visualized with non-UV light.

The degree of Target DNA fragmentation is proportional to the amount ofTransposome added over the 12.5-fold dilution of the 12.5 μM Transposomestock. At high concentrations of Transposome, the majority of the DNAfragments migrated in the gel at sizes less than 1000 by (FIG. 5, lanes3 and 9). At low concentrations of transposomes, the DNA fragmentsmigrated in the gel predominantly between 500 by and 6000 by (FIG. 5,lanes 8 and 14). Block arrow indicates migration free transposon endcomposition in gel.

Example 5 Target DNA Fragmentation and 5′-Tagging at 55° C. and 37° C.in the Presence of Dimethylformamide

To test the effect dimethylformamide on target DNA fragmentation and5′-tagging, HeLa genomic DNA was fragmented and tagged with MEtransposomes or A/B transposomes as follows.

“ME Transposomes” were formed by pre-incubating 12.5 μM TSase with 12.5μM MEDS transposon end compositions for 60 minutes at 37° C.

Duplicate reactions were set-up as follows:

TMgCl- Reagent TA TA-DMF TMgCl DMF 10X TA Reaction Buffer 2 μl — — — 5XTA-DMF Reaction Buffer — 4 μl — — 10X TMgCl Reaction Buffer — — 2 μl —5X TMgCl-DMF Reaction — — — 4 μl Buffer 50 ng/μl HeLa Genomic DNA 1 μl 1μl 1 μl 1 μl ME Transposome (12.5 μM) 1 μl 1 μl 1 μl 1 μl water 16 μl 14 μl  16 μl  14 μl  Final Volume: 20 μl  20 μl  20 μl  20 μl 

“A-MEDS Transposomes” and “B-MEDS Transposomes” were formed bypre-incubating 12.5 μM TSase with 12.5 μM A-MEDS or 12.5 μM B-MEDStransposon end compositions, respectively, for 60 minutes at 37° C.A-MEDS and B-MEDS transposomes were combined in equal ratios to form“A/B Transposomes.”

Duplicate reactions were set-up as follows:

TMgCl- Reagent TA TA-DMF TMgCl DMF 10X TA Reaction Buffer 2 μl — — — 5XTA-DMF Reaction Buffer — 4 μl — — 10X TMgCl Reaction Buffer — — 2 μl —5X TMgCl-DMF Reaction — — — 4 μl Buffer 50 ng/μl HeLa Genomic DNA 1 μl 1μl 1 μl 1 μl MA/B Transposome (12.5 μM) 1 μl 1 μl 1 μl 1 μl water 16 μl 14 μl  16 μl  14 μl  Final Volume: 20 μl  20 μl  20 μl  20 μl 

Reactions were incubated for 5 minutes at 37° C. or for 5 minutes at 55°C. Reactions were stopped with 5 microliters of stop solution (15%sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma0-7252], and Proteinase K at 100 micrograms per ml), mixed, and heatedat 70° C. for 10 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. Gelswere stained with SYBR Gold and DNA was visualized with non-UV light.

Dimethylformamide improved the efficiency of the fragmentation and5′-tagging reaction as judged by the decrease in the reaction product MWdistribution (FIG. 6, compare lanes 4, 6, 8, 10, 13, 15, 17, and 19 tolanes 3, 5, 7, 9, 12, 14, 16, and 18, respectively). Similarly,reactions in the presence of TMgCl reaction buffer were more efficientthan reactions in the presence of TA reaction buffer. Finally, reactionsat 55° C. appeared to improve the overall efficiency of the reactionscompared to reactions at 37° C. (FIG. 6, compare lanes 4-10 to lanes12-19). The block arrow indicates the migration of free transposon endcomposition in the gel.

Example 6 Fragmentation Tagging of Target DNA Using MuA Transposase

HyperMu™ MuA transposase (EPICENTRE) at a final concentration of 1 unitper microliter, and then a range of MuA transposase proteinconcentrations between about 0.01 micrograms and about 0.5 micrograms ofprotein per microliter of reaction mixture, was incubated in a50-microliter reaction containing MuA transposase reaction buffer(EPICENTRE), 1 microgram of phage T7 D111 target DNA, and 1 micromolarof the pR₁R₂ MuA transposon end for 1 hour 37° C.

The reaction was stopped and products analyzed by agarose gelelectrophoresis as described in EXAMPLE 3.

Fragmentation of the phage T7 D111 target DNA was much less than wasobserved using the EZ-Tn5™ Tn5 transposase at all levels of MuAtransposase tested. A very small amount of fragmentation was observedonly with the highest concentration of MuA transposase tested. Thus, useof MuA transposase and the pR₁R₂ MuA transposon end was far lessefficient for fragmenting and 5′-tagging target DNA than the EZ-Tn5™.Tn5 hyperactive transposase and EZ-Tn5™ Tn5 ME transposon end.

Example 7 Tagging the 3′-Ends of the 5′-Tagged DNA Fragments

A. Two-Primer PCR

In order to tag the 3′ ends of the transposition-generated and 5′-taggedDNA fragments with the transferred transposon end sequence, thefollowing reaction is carried out:

-   -   22 microliters 0.5-1 Kbp size-selected 5′-tagged transposition        products    -   25 microliters Failsafe™ PCR PreMix C    -   1 microliter Failsafe™ DNA polymerase (EPICENTRE)    -   2 microliters 5-micromolar of each PCR oligonucleotide primer,        of which one is complementary to the 5′-portion of each of the        5′-portions of the two different transferred transposon ends.    -   50 microliters total reaction volume

Since FailSafe DNA polymerase has strand-displacement and 5′ nucleaseactivity The polymerization of the method is carried out by incubatingthe reaction for 10 minutes at 70° C. (3′ DNA polymerase extensionstep), thereby generating 5′- and 3′-tagged DNA fragments (FIG. 8).

Then, the reaction is incubated at 94° C. for 5 minutes to denature theDNA.

Amplifying the 5′- and 3′-tagged DNA fragments is performed by PCRamplifying the 5′- and 3′-tagged DNA fragments using the two PCR primerseach of which is complementary to the 5′-portion of one of the twodifferent transferred transposon ends.

The PCR reaction mix above is subjected to PCR for 20 cycles with thefollowing cycling conditions:

-   -   94° C. 10 sec.    -   55° C. 10 sec.    -   72° C. 2 min.

Gel analysis indicated that the PCR products of the expected size range(0.5-1 Kbp) were produced.

Control reactions are also carried out: If size-selected transpositionproducts are heat denatured prior to PCR and with no 3′ DNA polymeraseextension step, no 0.5-1 Kbp PCR products are produced.

B. Single Primer PCR

In order to tag the 3′ ends of the transposition-generated and 5′ME-tagged fragments with the ME sequence, the following reaction wascarried out:

23 microliters 0.5-1 Kbp size-selected 5′-tagged transposition products25 microliters Failsafe ™ PCR PreMix C 1 microliter Failsafe ™ DNApolymerase (EPICENTRE) 1 microliter 5-micromolar pMETS as a PCRoligonucleotide primer 50 microliters

Since FailSafe DNA polymerase has strand-displacement and 5′ nucleaseactivity, the method was carried out by incubating the reaction for 10minutes at 70° C. (3′ DNA polymerase extension step), thereby generating5′- and 3′-tagged DNA fragments (FIG. 7).

Then, the reaction was incubated at 94° C. for 5 minutes to denature theDNA.

Amplifying the 5′- and 3′-tagged DNA fragments was performed by PCRamplifying the 5′- and 3′-tagged DNA fragments using the pMETs as theonly PCR oligonucleotide primer. The PCR reaction mix above wassubjected to PCR for 20 cycles with the following cycling conditions:

-   -   94° C. 10 sec.    -   55° C. 10 sec.    -   72° C. 2 min.

Gel analysis indicated that the PCR products of the expected size range(0.5-1 Kbp) were produced.

Control reactions were also carried out: If size-selected transpositionproducts were heat denatured prior to PCR and with no 3′ DNA polymeraseextension step, no 0.5-1 Kbp PCR products were produced.

Example 8 Amplification and Deep Sequencing of a DNA Fragment Library

In order to generate a non-selective DNA fragment library that can beamplified prior to library preparation, DNA fragments were generated andtagged at the 3′-end and the 5′-end using “ME Transposomes”.

“ME Transposomes” were formed by pre-incubating 10 μM TSase with 10 μMMEDS transposon end compositions for 10 minutes on ice.

A 43 kb cosmid DNA was fragmented and 5′-tagged in the followingreaction:

Reagent Volume 10X TA Reaction Buffer 5 μl 142 ng/μl 43 kb Cosmid DNA 7μl ME Transposome (10 μM) 5 μl water 33 μl Final Volume: 50 μl

The reaction was incubated 2 hours minutes at 37° C. An additional 5 μlof 10 μM ME Transposome was added to the reaction and incubated anadditional 2 hours at 37° C.

In order to tag the 3′ ends of the transposition-generated and 5′-taggedDNA fragments with the transferred transposon end sequence, the reactionproducts were incubated with a strand-displacing polymerase mix(FailSafe™) and dNTPs.

A portion of the transposition-generated and 5′-tagged DNA fragments wasdiluted 1:10 prior to 3′-end tagging and amplification to characterizeamplification of 4 ng of DNA library template. In order tonon-selectively amplify the entire population of tagged DNA fragmentsusing a single primer PCR, the following reaction was performed with theMETS PCR primer, which hybridizes only to the transposon end sequenceand does not contain additional 3′ sequence information.

Reagent Volume 2X FailSafe™ PCR Buffer E 12.5 μl 5′-tagged DNA Fragments(diluted 1:10)  2 μl METS PCR Primer (25 μM)  1 μl FailSafe™ PCR Enzyme,2.5 U/μl  1 μl water  8.5 μl Final Volume: 25 μl

The reaction was incubated as follows:

-   -   72° C./2:00* *—In order to tag the 3′ ends of the        transposition-generated and 5′-tagged DNA fragments with the        transferred transposon end sequence, the reaction products were        incubated with a strand-displacing polymerase mix (FailSafe™)        and dNTPs prior to the denaturation step (See FIG. 7).    -   98° C./1:00    -   25 cycles of (98° C./0:10, 55° C./0:10, 72° C./1:00)    -   4° C. hold

The amplified and unamplified reaction products were purified using aQIAGEN PCR-Clean-up column per the manufacturer's instructions and usedas input for step 3.4 of the standard Roche/454 FLX library preparationprotocol per the manufacturer's instructions (USM00048.A, October 2008).

Deep sequencing of the transposon-fragmented libraries produced a singlecontig of the expected size with read length, accuracy, and coveragethat was comparable to a control library produced using nebulization(FIG. 9). These data are consistent with the non-selective and massivelyparallel amplification of a DNA fragment library.

Example 9 Preparation of Bar Coded Roche/454 FLX-Compatible SequencingLibraries by Adding Additional 5′ and 3′ Sequencing Tag InformationUsing PCR with Adaptor Oligonucleotides

In order to generate a bar coded DNA fragment library that can be useddirectly in emPCR for 454 GS FLX sequencing, lambda genomic DNA wasfragmented and 5′-tagged with ME transposomes. Non-selective adaptoroligonucleotides were used during PCR to append the DNA library with 454FLX emPCR and sequencing adaptor and barcode sequence (FIG. 10).

“ME Transposomes” were formed by pre-incubating 12.5 μM TSase with 12.5μM MEDS transposon end compositions for 60 minutes at 37° C.

Lambda genomic DNA was fragmented and 5′-tagged in the followingreaction:

Reagent Volume 10X TA Reaction Buffer 5 μl 500 ng/μl Lambda DNA 2 μl METransposome (12.5 μM) 2 μl water 39 μl Final Volume: 48 μl

The reaction was incubated 2 hours at 37° C. An additional 2 μl of 12.5μM ME Transposome was added to the reaction and incubated an additional2 hours at 37° C.

The reaction products were purified using QIAGEN PCR-Clean-Up column perthe manufacturer's instructions.

In order to non-selectively amplify and append the DNA fragment librarywith adaptors compatible with Roche/454 FLX emPCR and sequencing, PCRwas performed using adaptor oligos which hybridize to the transposon endsequence and do not contain additional 3′ sequence information (FIG.10).

Reagent Volume 2X FailSafe™ PCR Buffer E 25   μl 5′-tagged DNA Fragments(20 ng/μl)  0.5 μl A-MID2-METS PCR Primer (2.5 μM)  1 μl B-METS PCRPrimer (2.5 μM)  1 μl FLX-A PCR Primer (50 μM)  1 μl FLX-B PCR Primer(50 μM)  1 μl FailSafe™ PCR Enzyme, 2.5 U/μl  1 μl water 24.5 μl FinalVolume: 50 μl

The reaction was incubated as follows:

-   -   72° C./5:00* *—In order to tag the 3′ ends of the        transposition-generated and 5′-tagged DNA fragments with the        transferred transposon end sequence, the reaction products were        incubated with a strand-displacing polymerase mix (FailSafe™)        and dNTPs prior to the denaturation step (See FIG. 7).    -   98° C./2:00    -   4 cycles of (98° C./0:10, 37° C./0:30, 72° C./3:00)    -   6 cycles of (98° C./0:10, 64° C./3:00)    -   4° C. hold

Control reactions omitted the A-MID2-METS and B-METS PCR primers andcontained 20 ng of 5′-tagged DNA Fragments.

The PCR reaction produced an emPCR-compatible library with the expectedMW distribution and was similar to that of the transposition-generatedand 5′-tagged DNA fragments (FIG. 11, lanes 3 and 4). The lack ofdetectable amplification products in reactions lacking the adaptorprimers (A-MID2-METS and B-METS) is consistent with specificamplification of a FLX-A and FLX-B-tagged DNA library (FIG. 11, lane 5).

Example 10 Deep Sequencing of Roche/454 FLX Titanium-CompatibleSequencing Library from 50 ng of Viral Amplicon cDNA

In order to generate a non-selective DNA fragment library that can beused directly in emPCR for Roche/454 FLX Titanium sequencing, ampliconDNA was fragmented and 5′-tagged with ME transposomes. Non-selectiveadaptor oligonucleotides were used to append the DNA library withRoche/454 FLX Titanium emPCR and sequencing adaptor sequence (FIG. 10).

“ME Transposomes” were formed by pre-incubating 12.5 μM TSase with 12.5μM MEDS transposon end compositions for 60 minutes at 37° C. This stockwas diluted to 7.5 μM in TSase storage buffer (50 mM Tris chloride pH7.5, 50% glycerol, 0.1 mM EDTA, 1 mM DTT, 500 mM Sodium chloride, 0.5%v/v NP-40, 0.5% v/v Tween-20).

Viral amplicon cDNA was fragmented and 5′-tagged in the followingreaction:

Reagent Volume 10X TA Reaction Buffer 5 μl 9.4 ng/μl Viral Amplicon cDNA5.5 μl ME Transposome (7.5 μM) 1 μl water 38.5 μl Final Volume: 50 μl

The reaction was incubated 15 minutes at 55° C. An additional 1 μl of7.5 μM ME Transposome was added to the reaction and incubated anadditional 15 minutes at 55° C.

The reaction products were purified using QIAGEN PCR-Clean-Up column perthe manufacturer's instructions using two 11 μL elutions that werepooled.

In order to non-selectively amplify and append the DNA fragment librarywith adaptors compatible with Roche/454 FLX Titanium emPCR andsequencing, PCR was performed using adaptor oligos which hybridize tothe transposon end sequence and do not contain additional 3′ sequenceinformation (FIG. 10).

Reagent Volume 2X FailSafe™ PCR Buffer E 25 μl 5′-tagged DNA Fragments(~20 μl recovered) 5 μl Ti A-METS PCR Primer (0.5 μM) 1 μl Ti B-METS PCRPrimer (0.5 μM) 1 μl Ti A PCR Primer (10 μM) 1 μl Ti B PCR Primer (10μM) 1 μl FailSafe™ PCR Enzyme, 2.5 U/μl 1 μl water 15 μl Final Volume:50 μl

The reaction was incubated as follows:

-   -   72° C./5:00* *—In order to tag the 3′ ends of the        transposition-generated and 5′-tagged DNA fragments with the        transferred transposon end sequence, the reaction products were        incubated with a strand-displacing polymerase mix (FailSafe™)        and dNTPs prior to the denaturation step (See FIG. 7).    -   98° C./2:00    -   4 cycles of (98° C./0:10, 55° C./0:30, 72° C./3:00)    -   6 cycles of (98° C./0:10, 64° C./3:00)    -   4° C. hold

The PCR reaction produced an emPCR-compatible library with the expectedMW distribution and was similar to that of the transposition-generatedand 5′-tagged DNA fragments (FIG. 12, lanes 2 and 3).

Deep sequencing of the library on ⅛^(th) of a Roche/454 FLX Titaniumplate provided ˜80,000 reads with ˜95% of the reads mapping to thereference viral genome yield with the expected coverage (data notshown). These data are consistent with the non-selective and massivelyparallel amplification of a DNA fragment library.

Example 11 Preparation of Bar Coded Illumina GAII-Compatible SequencingLibraries by Adding Additional 5′ and 3′ Sequencing Tag InformationUsing PCR with Adaptor Oligonucleotides

In order to generate a DNA fragment library that can be used directly inbPCR for Illumina GAII sequencing, lambda genomic DNA was fragmented and5′-tagged with A/B transposomes. Non-selective adaptor oligonucleotideswere used to append the DNA library with Illumina GAII bPCR adaptor andbarcode sequence (FIG. 13).

“A-MEDS Transposomes” and “B-MEDS Transposomes” were formed bypre-incubating 12.5 μM TSase with 12.5 μM A-MEDS or 12.5 μM B-MEDStransposon end compositions, respectively, for 60 minutes at 37° C.

Lambda genomic DNA was fragmented and 5′-tagged in the followingreaction:

Reagent Volume 10X TA Reaction Buffer 5 μl 500 ng/μl Lambda DNA 2 μlA-MEDS Transposome (12.5 μM) 2 μl B-MEDS Transposome (12.5 μM) 2 μlwater 39 μl Final Volume: 50 μl

The reaction was incubated 2 hours at 37° C. An additional 2 μl of 12.5μM A-MEDS Transposome and 2 μl of 12.5 μM B-MEDS Transposome was addedto the reaction and incubated an additional 2 hours at 37° C.

The reaction products were purified using QIAGEN PCR-Clean-Up column perthe manufacturer's instructions.

In order to non-selectively amplify and append the DNA fragment librarywith adaptors compatible with Illumina GAII bridge PCR and sequencing,PCR was performed using adaptor oligos which hybridize to the transposonend sequence and do not contain additional 3′ sequence information (FIG.13).

Reagent Volume 2X FailSafe™ PCR Buffer E 25 μl 5′-tagged DNA Fragments(20 ng/μl)  0.5 μl BP1-A PCR Primer (0.5 μM)  1 μl BP2-ID 1-B PCR Primer(0.5 μM)  1 μl BP1 PCR Primer (10 μM)  1 μl BP2 PCR Primer (10 μM)  1 μlFailSafe™·PCR Enzyme, 2.5 U/μl 1 μl water 24.5 μl Final Volume: 50 μl

The reaction was incubated as follows:

-   -   72° C./5:00* *—In order to tag the 3′ ends of the        transposition-generated and 5′-tagged DNA fragments with the        transferred transposon end sequence, the reaction products were        incubated with a strand-displacing polymerase mix (FailSafe™)        and dNTPs prior to the denaturation step (See FIG. 8).    -   98° C./2:00    -   10 cycles of (98° C./0:10, 58° C./0:30, 72° C./3:00)    -   4° C. hold

Control reactions omitted the BP1-A and BP2-ID1-B PCR primers andcontained 20 ng of 5′-tagged DNA Fragments.

The PCR reaction produced an bPCR-compatible library with the expectedMW distribution and was similar to that of the transposition-generatedand 5′-tagged DNA fragments (FIG. 14, lanes 3 and 4). The lack ofdetectable amplification products in reactions lacking the adaptorprimers (BP1-A and BP2-ID1-B) is consistent with specific amplificationof a BP1- and BP2-tagged DNA library FIG. 14, lane 5).

Example 12 Deep Sequencing of an Illumina GAII-Compatible SequencingLibrary

In order to generate a non-selective DNA fragment library that can beused directly in bPCR for Illumina GAII sequencing, E. coli CC118genomic DNA was fragmented and 5′-tagged with A/B transposomes.Non-selective adaptor oligonucleotides were used to append the DNAlibrary with Illumina GAII bPCR adaptors (FIG. 13).

E. coli CC118 genomic DNA was fragmented, 5′-tagged and purified asdescribed in EXAMPLE 1.

In order to non-selectively amplify and append the DNA fragment librarywith adaptors compatible with Illumina GAII bridge PCR and sequencing,PCR was performed using adaptor oligos which hybridize to the transposonend sequence and do not contain additional 3′ sequence information.

Reagent Volume 2X FailSafe™ PCR Buffer E 25 μl 5′-tagged DNA Fragments(20 ng/μl) 0.5 μl BP1-A PCR Primer (0.5 μM)  1 μl BP2-B PCR Primer (0.5μM)  1 μl BP1 PCR Primer (10 μM)  1 μl BP2 PCR Primer (10 μM)  1 μlFailSafe™ PCR Enyme, 2.5 U/μl  1 μl water 24.5 μl Final Volume: 50 μl

The reaction was incubated as follows:

-   -   72° C./5:00* *—In order to tag the 3′ ends of the        transposition-generated and 5′-tagged DNA fragments with the        transferred transposon end sequence, the reaction products were        incubated with a strand-displacing polymerase mix (FailSafe™)        and dNTPs prior to the denaturation step (See FIG. 8).    -   98° C./2:00    -   10 cycles of (98° C./0:10, 58° C./0:30, 72° C./3:00)    -   4° C. hold

Deep sequencing of the generated library achieved coverage of the ˜4.6Mb genome with an average depth of ˜115× (data not shown). These dataare consistent with the non-selective and massively parallelamplification of a DNA fragment library that is compatible withRoche/454 FLX Titanium emPCR and sequencing.

Example 13 Comparison of Prior Art Methods to Embodiments of the PresentInvention and Protocols for Kits

The workflow and timeline for preparation of a tagged DNA fragmentlibrary by the methods of the present invention is compared to theworkflow and timeline for preparation of such libraries by the currentmethods typically used. A table comparing the process steps and the timerequired at each step is shown in FIG. 15. The methods of the presentinvention require fewer steps, less hands-on time, and less timeoverall.

Example 14 In Vitro Transposition-Mediated DNA Fragmentation and TaggingUsing EZ-Tn5™ Transposase and a Hairpin EZ-Tn5™ Transposon EndComposition

The following components were mixed to form Hairpin Transposomes™ (i.e.,the hairpin transposon end composition complex with the transposase) ata final concentration of 25 micromolar (See FIG. 16):

10 microliters EZ-Tn5 ™ Hairpin transposon end composition (250micromolar) 27 microliters EZ-Tn5 ™ Transposase (91.4 micromolar) 63microliters Transposase Storage Buffer 100 microliters 

The following reaction mixture was assembled:

x microliters water to a final volume of 50 microliters 5 microliters10X EZ-Tn5 ™ Transposition Buffer 1 microgram target DNA in 1 to 40microliters 0.1, 2, 4, or 6 microliters 25 micromolar HairpinTransposomes ™ 50 microliters

After mixing, the reaction was incubated for 2 hours at 37° C. Thereaction was stopped with and equal volume of stop solution (15%sucrose, 66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma0-7252]), mixed, and heated at 70° C. for 10 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. Gelswere stained with SYBR Gold and DNA was visualized with non-UV light.

Fragmentation of Target DNA is proportional to the amount of addedTransposome (FIG. 17).

Example 15 Tagged Circular DNA Fragments are Resistant to T5 Exonuclease

5′-Tagged DNA fragments from EXAMPLE 14 were isolated using PCR Clean-upNucleoTraP®CR (Macherey-Nagel, GmbH) per the manufacture instructions.In order to create tagged circular DNA fragments, the recovered DNA wasincubated with a non-strand displacing polymerase lacking 5′-to-3′exonuclease activity (e.g. T4 DNA polymerase) and a template-dependentligase (e.g. E. coli ligase) in the presence of dNTPS and β-NAD.

The following reaction mixture was assembled:

14 microliters 5′-Tagged DNA Fragments from Example 1  2 microliters 10XEZ-Tn5™ Transposition Buffer  1 microliter 2 mM β-NAD  1 microliter 4 mMdNTPs  1 microliter E. coli DNA Ligase (10 U/μl)  1 microliter 14 DNAPolymerase (0.5 U/μl) 20 microliters

The reaction was incubated for 15 minutes at ambient temperature.Reactions were stopped by incubating 20 minutes at 75° C. A portion ofthe reaction (10 μl) was incubated with 10 units of T5 Exonuclease for 5minutes at 37° C. to degrade linear (non-circularized) DNA fragments.

All reactions were treated with 2 μl of stop solution (15% sucrose, 66mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma 0-7252]),mixed, and heated at 70° C. for 5 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. Gelswere stained with SYBR Gold and DNA was visualized with non-UV light.

Treatment with T4 DNA polymerase and E. coli ligase converted a portionof the DNA fragments to T5 exonuclease-resistant tagged circular DNAfragment molecules that are readily detected (FIG. 18, lane 9).Moreover, the molecular weight distribution of the T5exonuclease-resistant DNA is comparable to the input DNA, demonstratinga lack of molecular weight bias in this reaction.

Control reactions were also carried out. When the DNA fragments were nottreated, were treated with T4 DNA polymerase alone, or treated with E.coli ligase alone, T5 exonuclease-resistant tagged circular DNAfragments were not detected (FIG. 18; lanes 3, 5, and 7).

Example 16 Tagging the 3′-Ends of the Transposition-Generated 5′-TaggedDNA Fragments Using a Nucleic Acid Ligase and a Ligation TaggingOligonucleotide

5′-tagged fragmented DNA from the sizing procedure above (Example 1) wasused for tagging the 3′ ends of the 5′-tagged DNA fragments using anucleic acid ligase and a ligation tagging oligonucleotide (FIG. 21).

In order to tag the 3′ ends of the transposition-generated 5′-tagged DNAfragments with a second tag comprising the Roche 454 sequencing tag(4N454B), the following reaction was carried out.

43 microliters 0.5-1-Kb size-selected 5′-tagged DNA fragments fromEXAMPLE 1 5 micro liters 10X Ligase Reaction Buffer (0.2 M Tris-HCl pH8.3, 100 mM MgCl₂, 250 mM KCl, 5 mM β-NAD) 1 micromolar 4N454Boligonucleotide (5′pNNNNCTGAGCGGGCTGGCAAGGC 3′(SEQ ID NO: 6)) 1microliter E. coli DNA Ligase (10 units per microliter)

After mixing, the reaction was incubated for 1 hour at room temperature.Then, the reaction was stopped, the ligase was inactivated, and the DNAwas denatured by incubation at 95° C. for 5 min. Then, the reaction waschilled immediately in an ice water bath.

PCR analysis was used to show that the 5′ ends of the DNA fragmentsexhibited the transferred transposon end sequence of the EZ-Tn5transposon end and the 3′ ends exhibited the Roche 454 sequencing tag(4N454B).

The following PCR reaction was carried out as follows:

21 microliters water  1 microliter dual-tagged 5′- and 3′-tagged DNAfragments  1 microliter 5-micromolar PCR Primer 1 (pMETS)  1 microliter5-micromolar PCR Primer 2 (5′ATA GGC GCG CCG CCT TGC CAG CCC GCT CAG 3′0(SEQ ID NO: 21)  1 microliter FailSafe™ DNA polymerase 25 microlitersFailSafe™ 2X PCR PreMix C 50 microliters

PCR was carried out for 20 cycles, under the following conditions:

94° C. 10 sec.

55° C. 10 sec.

72° C. 2 min.

Gel analysis indicated that PCR products of the expected size range of0.5-1.0 KB were produced (data not shown).

Control reactions were also carried out. When the ligation reaction wascarried out without the ligase or without the Roche 454 sequencing tag(4N454B), no PCR products were produced. When either the pMETS PCRPrimer 1 or PCR Primer 2 was omitted from the PCR reaction, no 0.5-1-KBproducts were produced. When a ligation reaction was carried out withoutthe ligase or without the Roche 454 sequencing tag (4N454B), no PCRproducts were produced. When either the pMETS PCR Primer 1 or PCR Primer2 was omitted from the PCR reaction, no 0.5-1-KB products were produced(data not shown).

Example 17 Circularization of Tagged ssDNA Fragments from In VitroTransposition-Mediated DNA Fragmentation and 5′-Tagging Using theP454.1MEDS Transposon End Composition and EZ-Tn5™ Transposase

T7 D111 genomic DNA was 5′-end tagged and fragmented using p454MEDSEZ-Tn5™ transposon end composition in the following reaction:

x water to a final volume of 50 microliters 5 microliters 10X EZ-Tn5 ™Transposition Buffer 1 microgram target DNA in 1 to 40 microliters 2microliters p454.1MEDS transposon end composition (25 μM) 2 microlitersEZ-Tn5 ™ Transposase (at 10 units per microliter) 50 microliters Finalreaction volume

After mixing, the reaction was incubated for 1 hour at 37° C. Then, thereaction was stopped with 10 microliters of stop solution (15% sucrose,66 mM EDTA, 20 mM TRIS, pH 8.0, 0.1% SDS, 0.9% Orange G [Sigma 0-7252],and Proteinase K at 100 micrograms per ml), mixed, and heated at 50° C.for 10 minutes.

DNA was analyzed by 1% agarose gel electrophoresis in TAE buffer. Gelswere stained with SYBR Gold and DNA was visualized with non-UV light.

The target DNA was fragmented to a similar extent and to a similar sizerange as described in Example 3, using comparable quantities andconcentrations of the EZ-Tn5™ transposase and the pMEDS transposon end(FIG. 22B, lane 5). These data indicate that extending the pMETS oligowith the additional Roche 454 sequencing tag does not significantlyalter the efficiency of DNA fragmentation and tagging by EZ-Tn5™transposase. Omitting either the p454.1MEDS transposon end compositionor the EZ-Tn5™ transposase did not result in detectable DNAfragmentation (FIG. 22, lanes 3 and 4).

The 5′-tagged fragmented DNA from FIG. 22, lane 5 was heat-denatured andcircularized using template-independent ligase (See FIG. 22A) in thefollowing reaction:

x water to a final volume of 20 microliters 1 microliter 330 mMTris-acetate pH 7.8, 660 mM KOAc 1 microliter 50 mM MnCl₂ 4 microliters5 M Betaine 10 microliters 20 μg/ml denatured 5′-tagged fragmented DNA 4microliters 100 U/μl CIRCLIGASE ™ ssDNA Ligase (EPICENTRE) 20microliters Final reaction volume

The reaction was incubated 2 hours at 60° C. Then, the reaction productswere treated with 18 units of Exo I and 20 units of Exo III for 1 hourat 37° C. to eliminate non-circularized, linear DNA.

Example 18 PCR Analysis of Tagged Circular ssDNA

PCR analysis was performed using pMETS and pc454.1 as primers todemonstrate circularization; only ligated, circular ssDNA can beamplified to generate a linear dsDNA product that corresponds to thesize of the circular ssDNA. The PCR reaction was carried out as follows:

21 microliters water 1 microliter Exonuclease-treated CircLigasereaction (1:1000) 1 microliter 5 μM pMETS oligonucleotide as a primer 1microliter 5 μM pc454.1 oligonucleotide as a primer 1 microliterFailSafe ™ DNA polymerase 25 microliters FailSafe ™ 2X PCR PreMix C 50microliters Final reaction volume

PCR was carried out for 29 cycles, under the following conditions:

94° C. 10 sec.

50° C. 10 sec.

72° C. 1 min.

Gel analysis indicated that the size range of the produced PCR productswere comparable to 5′-tagged fragmented DNA (FIG. 23).

Control reactions were also carried out. When CIRCLIGASE™ ssDNA Ligasewas omitted from the ligation reaction, PCR products were generated thatindicated circularization of the p454.1METS transferred strand, but notthe 5′-tagged linear ssDNA fragments (FIG. 23, lane 1). When either thepMETS oligonucleotide (as a PCR primer) or the pc454.1 PCR Primer wasomitted from the PCR reaction, no products were produced (data notshown).

The fact that the PCR products had the same size distribution as the5′-tagged linear ssDNA fragments (compare FIG. 22, lane 5 and FIG. 23,lane 2) indicates that: 1) the p454.1MEDS transposon end composition canefficiently 5′-tag and fragment target DNA; 2) the annealedcomplementary 5′-tagged linear ssDNA fragments can be heat-denatured toyield denatured tagged linear ssDNA fragments that are substrates fortemplate-independent ligation; and 3) the 5′-tagged linear ssDNAfragments can be efficiently converted to tagged circular ssDNAfragments without a detectable bias (confirmed by PCR amplificationafter exonuclease I and exonuclease III treatment).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and systems of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A method for generating and sequencing a library of DNAfragments having first and second tag sequences, comprising: (a)providing a target DNA; (b) providing a plurality of transposomecomplexes, wherein (1) at least one transposome complex comprises ahyperactive Tn5 transposase and at least a first polynucleotidecomprising a Tn5-type transposon end sequence and a first tag sequence;and (2) at least one transposome complex comprises a hyperactive Tn5transposase and at least a second polynucleotide comprising a Tn5-typetransposon end sequence and a second tag sequence; wherein thetransposome complexes of (1) and (2) comprise different sequences; (c)incubating the target DNA and transposome complexes under conditionswhereby multiple transposon end sequences and associated tag sequencesare inserted into the target DNA and the target DNA is fragmented into amultitude of double-stranded DNA fragments comprising one of the firstor the second polynucleotides attached to each 5′ end of the DNAfragments; (d) non-selectively amplifying the double-stranded DNAfragments; and (e) sequencing the DNA fragments simultaneously in asingle multiplex format.
 2. The method of claim 1, wherein thetransposome complex of (1) comprises two of the first polynucleotides,and the transposome complex of (2) comprises two of the secondpolynucleotides.
 3. The method of claim 1, wherein a transposome complexcomprises a single polynucleotide that forms a hairpin.
 4. The method ofclaim 3, wherein the hairpin comprises a cleavable site, and (c) furthercomprises cleaving the cleavable sites of a DNA fragment, therebygenerating a DNA fragment having a fantail.
 5. The method of claim 1,wherein (c) further comprises incubating the DNA fragments with astrand-displacing DNA polymerase under conditions whereby 3′ ends of theDNA fragments are extended.
 6. The method of claim 1, wherein (c)further comprises incubating the library of DNA fragments with a DNApolymerase having 5′-to-3′ exonuclease activity, under conditionswhereby 3′ ends of the DNA fragments are extended.
 7. The method ofclaim 1, wherein (c) further comprises incubating the library of DNAfragments with a ligase and a DNA polymerase lacking bothstrand-displacing and 5′-to-3′ exonuclease activities, under conditionswhereby single-stranded gaps in the DNA fragments are filled in.
 8. Themethod of claim 1, wherein (c) further comprises incubating the libraryof DNA fragments with a ligase and a ligation oligonucleotide, underconditions whereby single-stranded gaps in the DNA fragments are filledin.
 9. The method of claim 1, wherein at least one tag sequencecomprises a first sequencing tag, and (e) further comprises contacting afirst sequencing primer comprising a portion corresponding to the firstsequencing tag with the DNA fragments.
 10. The method of claim 9,wherein a tag sequence comprises a second sequencing tag and (e) furthercomprises contacting a second sequencing primer comprising a portioncorresponding to the second sequencing tag with the DNA fragments. 11.The method of claim 1, wherein at least one tag sequence comprises afirst amplification tag, and (d) comprises contacting a DNA polymeraseand a first primer having a portion corresponding to the firstamplification tag with the DNA fragments.
 12. The method of claim 11,wherein a tag sequence comprises a second amplification tag, and (d)comprises contacting a second primer having a portion corresponding tothe second amplification tag with the DNA fragments.
 13. The method ofclaim 11, wherein the non-selectively amplifying is selected from thegroup consisting of a strand-displacement amplification reaction, arolling circle amplification reaction, a loop-mediated amplificationreaction, and PCR.
 14. The method of claim 1, wherein at least one tagsequence comprises a restriction site.
 15. The method of claim 1,wherein at least one tag sequence comprises a transcription promotersequence, and (d) comprises performing transcription-mediatedamplification with the DNA fragments.
 16. The method of claim 1, whereinat least one tag sequence comprises an address tag to identify sequencesfor each sample.
 17. The method of claim 1, wherein at least one tagsequence comprises a capture tag, and (e) further comprises providing asurface having an annealing site for the capture tag.
 18. The method ofclaim 17, wherein the capture tag is a first affinity binding moleculeand (e) further comprises providing a surface to which a second affinitybinding molecule is attached.
 19. The method of claim 1, wherein atleast one tag sequence comprises a detection tag.
 20. The method ofclaim 19, wherein the detection tag has a sequence that facilitatesdetection of the DNA fragment.
 21. The method of claim 1, wherein (e)further comprises capturing the DNA fragments on a surface.
 22. Themethod of claim 21, wherein the surface comprises at least a millionattached DNA fragments.
 23. The method of claim 21, wherein the surfaceis on a substrate selected from the group consisting of: a bead, a chip,a slide, a microtiter plate, a tube, a microchannel, and a dipstick. 24.The method of claim 21, wherein the surface comprises at least a millioncaptured DNA fragments.
 25. The method of claim 1, wherein (c) comprisesextending the 3′ ends of the DNA fragments.
 26. A method for generatingand sequencing a library of DNA fragments having first and second tagsequences, comprising: (a) providing a target DNA; (b) providing aplurality of transposome complexes, wherein (1) at least one transposomecomplex comprises a hyperactive Tn5 transposase and at least a firstpolynucleotide comprising a Tn5-type transposon end sequence and a firsttag sequence; and (2) at least one transposome complex comprises ahyperactive Tn5 transposase and at least a second polynucleotidecomprising a Tn5-type transposon end sequence and a second tag sequence;wherein the transposome complexes of (1) and (2) exhibit differentsequences; (c) incubating the target DNA and transposome complexes underconditions whereby multiple transposon ends and associated tag sequencesare inserted into the target DNA and the target DNA is fragmented into amultitude of double-stranded DNA fragments that exhibit one of the firstor the second tag sequences attached to each 5′ end of the DNAfragments; (d) joining a tag to the 3′ ends of the DNA fragments fromstep (c) to generate a library of di-tagged DNA fragments, eachexhibiting a 5′ first or second tag sequence and a 3′ tag sequence, (e)non-selectively amplifying the library of di-tagged DNA fragments; and(f) sequencing the di-tagged DNA fragments simultaneously in a singlemultiplex format.